Process for utilizing epitopes recognized by natural antibodies

ABSTRACT

The present invention relates to compositions and methods for the selection and use of surface exposed epitopes. The present invention includes in vivo and in vitro phage peptide diplay methods for the identification and selection of peptides and peptide associated factors with desired properties (e.g., targeting specificity, stability, etc.). The present invention further provides methods and compositions for the isolation and identification of peptide-specific antibodies. The present invention also includes methods and compositions employing nuclear localization signals for enhanced nuclear transport and expression of DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] (Provisional application Ser. No.) (Filing Date) 60/131,151 Apr.27, 1999 60/139,431 Jun. 7, 1999

FEDERALLY SPONSORED RESEARCH

[0002] This invention was made under a contract with an agency of theUnited States Government: U.S. Government agency: NIH Governmentcontract number: 5 R21 DK53314-02

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for theselection and use of surface exposed epitopes. The present inventionincludes in vivo and in vitro phage peptide display methods for theidentification and selection of peptides and peptide associated factorswith desired properties (e.g., targeting specificity, stability, etc.).The present invention further provides methods and compositions for theisolation and identification of peptide-specific antibodies and includesmethods and compositions employing nuclear localization signals forenhanced nuclear transport and expression of DNA.

BACKGROUND

[0004] Drug Delivery

[0005] A variety of methods and routes of administration have beendeveloped to deliver pharmaceuticals that include small molecular drugsand biologically-active compounds such as peptides, hormones, proteins,and enzymes to their site of action. Parenteral routes of administrationinclude intravascular (intravenous, intraarterial), intramuscular,intraparenchymal, intradermal, subdermal, subcutaneous, intratumor,intraperitoneal, and intralymphatic injections that use a syringe and aneedle or catheter. The blood circulatory system provides systemicspread of the pharmaceutical. Polyethylene glycol and other hydrophilicpolymers have provided protection of the pharmaceuticals in the bloodstream by preventing their interaction with blood components andincreasing the circulatory time of the pharmaceuticals throughpreventing opsonization, phagocytosis and uptake by thereticuloendothelial system. For example, the enzyme adenosine deaminasehas been covalently modified with polyethylene glycol to increase thecirculatory time and persistence of this enzyme in the treatment ofpatients with adenosine deaminase deficiency.

[0006] The controlled release of pharmaceuticals after theiradministration is under intensive development. Pharmaceuticals have alsobeen complexed with a variety of biologically-labile polymers to delaytheir release from depots. These polymers have included copolymers ofpoly(lactic/glycolic acid) (PLGA) (Jain, R. et al. Drug Dev. Ind. Pharm.24, 703-727 (1998), ethylvinyl acetate/polyvinyl alcohol (Metrikin, DCand Anand, R, Curr Opin Ophthalmol 5, 21-29, 1994) as typical examplesof biodegradable and non-degradable sustained release systemsrespectively.

[0007] Transdermal routes of administration have been effected bypatches and ionotophoresis. Other epithelial routes include oral, nasal,respiratory, and vaginal routes of administration. These routes haveattracted particular interest for the delivery of peptides, proteins,hormones, and cytokines which are typically administered by parenteralroutes using needles. For example, the delivery of insulin viarespiratory, oral, or nasal routes would be very attractive for patientswith diabetes mellitus. For oral routes, the acidity of the stomach (pHless than 2) is avoided for pH-sensitive compounds by concealingpeptidase-sensitive polypeptides inside pH-sensitive hydrogel matrix(copolymers of polyethyleneglycol and polyacrylic acid). After passinglow pH compartments of gastrointestinal tract such structures swell athigher pH releasing thus a bioactive compound (Lowman AM et al. J.Pharm. Sci. 88, 933-937 (1999). Capsules have also been developed thatrelease their contents within the small intestine based uponpH-dependent solubility of a polymer. Copolymers of polymethacrylic acid(Eudragit S, Rohm America) are known as polymers which are insoluble atlower pH but readily solubilized at higher pH, so they are used asenteric coatings (Z Hu et al. J. Drug Target., 7, 223, 1999).

[0008] Biologically active molecules may be assisted by a reversibleformation of covalent bonds. Quite often, it is found that the drugadministered to a patient is not the active form of the drug, but whatis a called a prodrug that changes into the actual biologically activecompound upon interactions with specific enzymes inside the body. Inparticular, anticancer drugs are quite toxic and are administered asprodrugs which do not become active until they come in contact with thecancerous cells (Sezaki, II., Takakura, Y., Hashida, M. Adv. Drug.Delivery Reviews 3, 193, 1989).

[0009] Liposomes were also used as drug delivery vehicles for lowmolecular weight drugs and macromolecules such as amphotericin B forsystemic fungal infections and candidiasis. Inclusion of anti-cancerdrugs such as adriamycin has been developed to increase their deliveryto tumors and reduce their access to other tissue sites (e.g. heart)thereby decreasing their toxicity. The use of pH-sensitive polymers inconjunction with liposomes represents another opportunity to modulatelipid bilayer permeability warranting thus triggered release ofencapsulated drugs. For example, hydrophobically-modifiedN-isopropylacrylamide-methacrylic acid copolymer can render regular eggPC liposomes pH-sensitive by pH-dependent interaction of graftedaliphatic chains with lipid bilayer (O Meyer et al., FEBS Lett., 421,61, 1998).

[0010] Gene And Nucleic Acid-Based Delivery

[0011] Gene or polynucleotide transfer is the cardinal process of genetherapy. The gene needs to be transferred across the cell membrane andenter the nucleus where the gene can be expressed. Gene transfer methodscurrently being explored include viral vectors and physical-chemicalmethods.

[0012] Viruses have evolved over millions of years to transfer theirgenes into mammalian cells. Viruses can be modified to carry a desiredgene and become a “vector” for gene therapy. Using standard recombinanttechniques, the harmful or superfluous viral genes can be removed andreplaced with the desired normal genes. This was first accomplished withmouse retroviruses. The development of retroviral vectors were thecatalyst that promoted current gene therapy efforts. However, theycannot infect all cell types very efficiently, especially in vivo. Otherviral vectors based on Herpes virus are being developed to enable moreefficient gene transfer into brain cells. Adenoviral and adenoassociatedvectors are being developed to infect lung and other types of cells.

[0013] Besides using viral vectors, it is possible to directly transfergenes into mammalian cells. Usually, the desired gene is placed withinbacterial plasmid DNA along with a mammalian promoter, enhancer, andother sequences that enable the gene to be expressed in mammalian cells.Several milligrams of the plasmid DNA containing all these sequences canbe prepared and purified from the bacterial cultures. The plasmid DNAcontaining the desired gene can be incorporated into lipid vesicles(liposomes including cationic lipids such as Lipofectin) that thentransfer the plasmid DNA into the target cell. Plasmid DNA can also becomplexed with proteins that target the plasmid DNA to specific tissues,just as certain proteins are taken up (endocytosed) by specific cells.Also, plasmid DNA can be complexed with polymers such as polylysine andpolyethylenimine. Another plasmid-based technique involves “shooting”the plasmid DNA on small gold beads into the cell using a “gun”.Finally, muscle cells in vivo have the unusual ability to take up andexpress plasmid DNA.

[0014] Gene therapy approaches can be classified into direct andindirect methods. Some of these gene transfer methods are most effectivewhen directly injected into a tissue space. Direct methods using many ofthe above gene transfer techniques are being used to target tumors,muscle, liver, lung, and brain. Other methods are most effective whenapplied to cells or tissues that have been removed from the body,genetically modified and then transplanted back into the body. Indirectapproaches in conjunction with retroviral vectors are being developed totransfer genes into bone marrow cells, lymphocytes, hepatocytes,myoblasts and skin cells.

[0015] Gene Therapy and Nucleic Acid-Based Therapies

[0016] Gene therapy is a revolutionary advance in the treatment ofdisease. It is an approach for treating disease which is different fromthe conventional surgical and pharmaceutical therapies. Conceptually,gene therapy is a relatively simple approach. If someone has a defectivegene, then gene therapy would fix the defective gene. The disease statewould be modified by manipulating genes instead of the gene products.Although, the initial motivation for gene therapy was the treatment ofgenetic disorders, it is becoming increasingly apparent that genetherapy will be useful for the treatment of a broad range of acquireddiseases such as cancer, infectious disorders (AIDS), heart disease,arthritis, and neurodegenerative disorders (Parkinsons and Alzheimers).

[0017] Gene therapy promises to take full-advantage of the majoradvances brought about by molecular biology. While biochemistry ismainly concerned with how the cell obtains the energy and matter that isrequired for normal function, molecular biology is mainly concerned withhow the cell gets the information to perform its functions. Molecularbiology wants to discover the flow of information in the cell. Using themetaphor of computers, the cell is the hardware while the genes are thesoftware. In this sense, the purpose of gene therapy is to provide thecell with a new program (genetic information) so as to reprogram adysfunctional cell to perform a normal function. The addition of a newcellular function is provided by the insertion of a foreign gene thatexpresses a foreign protein or a native protein at amounts that are notinitially present in the patient.

[0018] The inhibition of a cellular function is provided by anti-senseapproaches (that is acting against messenger RNA) and that includesusing oligonucleotides complementary to the messenger RNA sequence andribozymes. Messenger RNA (mRNA) is an intermediate in the expression ofthe DNA gene. The mRNA is translated into a protein. “Anti-sense”methods use a RNA sequence or an oligonucleotide that is madecomplementary to the target mRNA sequence and therefore bindsspecifically to the target messenger RNA. When this anti-sense sequencebinds to the target mRNA, the mRNA is somehow destroyed or blocked frombeing translated. Ribozymes destroy a specific mRNA by a differentmechanism. Ribozymes are RNA's that contain sequence complementary tothe target messenger RNA plus a RNA sequence that acts as an enzyme tocleave the messenger RNA, thus destroying it and preventing it frombeing translated. When these anti-sense or ribozyme sequences areintroduced into a cell, they would inactivate their specific target mRNAand reduce their disease-causing properties.

[0019] Several recessive genetic disorders are being considered for genetherapy. One of the first uses of gene therapy in humans has been usedfor the genetic deficiency of the adenosine deaminase (ADA) gene. Otherclinical gene therapy trials have been conducted for cystic fibrosis,familial hypercholesteremia caused by a defective LDL-receptor gene andpartial ornithine transcarbomylase deficiency. Both indirect and directgene therapy approaches are being developed for Duchenne musculardystrophy. Patients with this type of muscular dystrophy eventually diefrom loss of their respiratory muscles. Direct approaches include theintramuscular injection of naked plasmid DNA or adenoviral vectors.

[0020] A wide variety of gene therapy approaches for cancer are underinvestigation in animals and in human clinical trials. One approach isto express in lymphocytes and in the tumor cells cytokine genes thatstimulate the immune system to destroy the cancer cells. The cytokinegenes would be transferred into the lymphocytes by removing thelymphocytes from the body and infecting them with a retroviral vectorcarrying the cytokine gene. The tumor cells would be similarlygenetically modified by this indirect approach to express cytokineswithin the tumor. Direct approaches involving the expression ofcytokines in tumor cells in situ are also being considered. Other genesbesides cytokines may be able to induce an immune response against thecancer. One approach that has entered clinical trials is the directinjection of HLA-B7 gene (which encodes a potent immunogen) within lipidvesicles into malignant melanomas in order to induce a more effectiveimmune response against the cancer.

[0021] “Suicide” genes are genes that kill cells which express the gene.For example, the diphtheria toxin gene directly kill cells. The Herpesthymidine kinase (TK) gene kills cells in conjunction with acyclovir (adrug used to treat Herpes viral infections). Other gene therapyapproaches take advantage of our knowledge of oncogenes and suppressortumor genes—also known as anti-oncogenes. The loss of a functioninganti-oncogene plays a decisive role in childhood tumors such asretinoblastoma, osteosarcoma and Wilms tumor and may play an importantrole in more common tumors such as lung, colon and breast cancer.Introduction of the normal anti-oncogene back into these tumor cells mayconvert them back to normal cells. The activation of oncogenes alsoplays an important role in the development of cancers. Since theseoncogenes operate in a “dominant” fashion, treatment will requireinactivation of the abnormal oncogene. This can be done using either“anti-sense” or ribozyme methods that selectively inactivate a specificmessenger RNA in a cell.

[0022] Gene therapy can be used as a type of vaccination to preventinfectious diseases and cancer. When a foreign gene is transferred intoa cell and the protein is made, the foreign protein is presented to theimmune system differently from simply injecting the foreign protein intothe body. This different presentation is more likely to cause acell-mediated immune response which is important for fighting latentviral infections such as human immunodeficiency virus (HIV causes AIDS),Herpes and cytomegalovirus. Expression of the viral gene within a cellsimulates a viral infection and induces a more effective immune responseby fooling the body that the cell is actually infected by the virus,without the danger of an actual viral infection.

[0023] One direct approach uses the direct intramuscular injection ofnaked plasmid DNA to express a viral gene in muscle cells. The “gun” hasalso been shown to be effective at inducing an immune response byexpressing foreign genes in the skin. Other direct approaches involvingthe use of retroviral, vaccinia or adenoviral vectors are also beingdeveloped. An indirect approach has been developed to remove fibroblastsfrom the skin, infect them with a retroviral vector carrying a viralgene and transplant the cells back into the body. The envelope gene fromthe AIDS virus (HIV) is often used for these purposes. Many cancer cellsexpress specific genes that normal cells do not. Therefore, these genesspecifically expressed in cancer cells can be used for immunizationagainst cancer.

[0024] Besides the above immunization approaches, several other genetherapies are being developed for treating infectious disease. Most ofthese new approaches are being developed for HIV infection and AIDS.Many of them will involve the delivery of anti-sense or ribozymesequences directed against the particular viral messenger RNA. Theseanti-sense or ribozyme sequences will block the expression of specificviral genes and abort the viral infection without damaging the infectedcell. Another approach somewhat similar to the ant-sense approaches isto overexpress the target sequences for these regulatory HIV sequences.

[0025] Gene therapy efforts would be directed at lowering the riskfactors associated with atherosclerosis. Overexpression of the LDLreceptor gene would lower blood cholesterol in patients not only withfamilial hypercholesteremia but with other causes of high cholesterollevels. The genes encoding the proteins for HDL (“the good cholesterol”)could be expressed also in various tissues. This would raise HDL levelsand prevent atherosclerosis and heart attacks. Tissue plasminogenactivator (tPA) protein is being given to patients immediately aftertheir myocardial infarction to digest the blood clots and open up theblocked coronary blood vessels. The gene for tPA could be expressed inthe endothelial cells lining the coronary blood vessels and therebydeliver the tPA locally without providing tPA throughout the body.Another approach for coronary vessel disease is to express a gene in theheart that produces a protein that causes new blood vessels to grow.This would increase collateral blood flow and prevent a myocardialinfarction from occurring.

[0026] Neurodegenerative disorders such as Parkinson's and Alzheimer'sdiseases are good candidates for early attempts at gene therapy.Arthritis could also be treated by gene therapy. Several proteins andtheir genes (such as the IL-1 receptor antagonist protein) have recentlybeen discovered to be anti-inflammatory. Expression of these genes injoint (synovial) fluid would decrease the joint inflammation and treatthe arthritis.

[0027] In addition, methods are being developed to directly modify thesequence of target genes and chromosomal DNA. The delivery of a nucleicacid or other compound that modifies the genetic instruction (e.g., byhomologous recombination) can correct a mutated gene or mutate afunctioning gene.

[0028] Liver Gene Therapy

[0029] Liver is one of the most important target tissues for genetherapy given its central role in metabolism (e.g. lipoproteinmetabolism in various hypercholesterolemias) and the secretion ofcirculating proteins (e.g. clotting factors in hemophilia). At least onehundred different genetic disorders could be corrected by liver-directedgene therapy. Their cumulative frequency is approximately one percent ofall births. In addition, multifactorial disorders are also amenable toliver gene therapy. For example, diabetes mellitus could be treated byexpressing the insulin gene within hepatocytes whose physiology mayenable glucose-regulated insulin secretion. Acquired disorders such aschronic hepatitis (particularly important in Asia) could also be treatedby polynucleotide-based liver therapies.

[0030] A variety of techniques have been developed to transfer genesinto the liver. Jon Wolff and colleagues suggested the liver as a targettissue for gene therapy by demonstrating that primary hepatocytes couldbe efficiently infected with retroviral vectors (Wolff, et al., Proc.Natl. Acad. Sci. USA, 84:3444-3348. 1987). Cultured hepatocytes havebeen genetically modified by retroviral vectors and implanted back intolivers of animals and humans (Grossman, et al., Nature Genet.,6:335-341. 1994, Chowdhury, et al., Science, 254:1802-1805. 1991,Ledley, et al., Somat. Cell Mol. Genet., 13:145-54. 1987). Retroviralvectors have also been delivered directly to livers in which hepatocytedivision was induced by partial hepatectomy or cytokines (Bosch, et al.,Journal of Clinical Investigation, 98:2683-7. 1996, Ferry, et al., Proc.Natl. Acad. Sci. USA, 88:8377-8381. 1991, Kaleko, et al., Hum. GeneTher., 2:27-32. 1991, Kay, et al., Hum. Gene Ther., 3:641-7. 1992,Hafenrichter, et al., J. Surgical Res., 56:510-7. 1994, Rettinger, etal., Proc. Natl. Acad. Sci. USA, 91:1460-4. 1994). Injection ofadenoviral vectors into the portal or systemic circulatory systems leadsto high levels of foreign gene expression that is transient (Sullivan,et al., Human Gene Therapy, 8:1195-206. 1997, Jaffe, et al., NatureGenet., 1:372-378. 1992, Li, et al., Hum. Gene Ther., 4:403-409. 1993,Stratford-Perricaudet, et al., Hum. Gene Ther., 1:241-56. 1990).Long-term expression of AAV vectors or retroviral vectors derived fromlentiviruses has been recently reported for liver and muscle (Snyder, etal., Nature Genetics, 16:270-6. 1997, Herzog, et al., Nature Medicine,5:56-63. 1999, Linden and Woo, Nature Medicine, 5:21-22. 1999, Snyder,et al., Nature Medicine, 5:64-70. 1999) (Xiao, et al., Journal ofVirology, 70:8098-8108. 1996, Fisher, et al., Nature Medicine,3:306-312. 1997, Herzog, et al., Proceedings of the National Academy ofSciences of the United States of America, 94:5804-5809. 1997) (Kafri, etal., Nature Genetics, 17:214-317. 1997). Since AAV and retroviralvectors require administration directly in the liver or its bloodvessels, hepatocyte-targeting peptides would improve their utility.Adenoviral vectors can target hepatocytes after peripheral veininjection but hepatocyte targeting would improve their safety andefficacy. Several groups are developing approaches to modify thetargeting of retroviral and adenoviral vectors, that could readilyincorporate the peptides discovered in this proposal (Reynolds andCuriel, The Development of Human Gene Therapy. Editor: T. Friedmann,Cold Spring Harbor Press, pp. 111-130.1999).

[0031] Non-viral transfer methods have included polylysine complexes ofasialoglycoproteins that are systemically administered (Wu and Wu,Biochemistry, 27:887-92. 1988, Wu, et al., Journal of BiologicalChemistry, 264:16985-7. 1989, Wilson, et al., Journal of BiologicalChemistry, 267:963-7. 1992). Plasmid DNA expression in the liver hasalso been achieved via liposomes delivered by tail vein or intraportalroutes (Kaneda, et al., J. Biol. Chem., 264:12126-12129. 1989, Kaneda,et al., Science, 243:375-378. 1989, Soriano, et al., Proc. Natl. Acad.Sci. USA, 80:7128-7131. 1993). One lab has shown that high levels ofhepatocyte expression can be achieved by the injection of naked plasmidDNA (pDNA) into liver vessels or tail vein (Budker, et al., GeneTherapy, 3:593-8. 1996, Zhang, et al., Human Gene Therapy, 8:1763-72.1997).

[0032] Paradigm for Development of Vectors

[0033] The current paradigm for the development of non-viral and viralvectors is to incorporate in a combinatorial fashion functional groupsthat enable particular transfer steps. These functional groups,initially discovered within proteins and viruses, serve as signals or“addresses” that interact with cellular components and cause the proteinor virus to enter a particular sub-cellular compartment. These samesignals can be incorporated into non-viral or viral vectors to enhanceeach transport step required for the therapeutic genes to eventuallyenter the cellular nucleus where the gene expresses its therapeuticfunction. These signals include surface molecules that resistinactivation in the blood, maintaining their ability to direct thevector toward target cells. After particle binding to the cell, theparticle must contain other molecules to release the particle DNA intothe cytoplasm. Finally, other functional groups can enable cytoplasmictransport to the nuclear membrane and traversal of the nuclear pore intothe nucleus.

[0034] Polymers for Drug and Nucleic Acid Delivery

[0035] Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used in research for the delivery ofnucleic acids (polynucleotides and oligonucleotides) to cells with aneventual goal of providing therapeutic processes. Such processes havebeen termed gene therapy or anti-sense therapy. One of the severalmethods of nucleic acid delivery to the cells is the use of complexes.It has been shown that cationic proteins like histones and protamines orsynthetic polymers like polylysine, polyarginine, polyornithine, DEAEdextran, polybrene, and polyethylenimine may be effective intracellulardelivery agents while small polycations like spermine are ineffective.The following are some important principles involving the mechanism bywhich polycations facilitate uptake of DNA:

[0036] Polycations provide attachment of DNA to the cell surface. Thepolymer forms a cross-bridge between the polyanionic nucleic acids andthe polyanionic surfaces of the cells. As a result the main mechanism ofDNA translocation to the intracellular space might be non-specificadsorptive endocytosis which may be more effective then liquidendocytosis or receptor-mediated endocytosis. Furthermore, polycationsare a convenient linker for attaching specific receptors to DNA and asresult, DNA-polycation complexes can be targeted to specific cell types.

[0037] Polycations protect DNA in complexes against nucleasedegradation. This is important for both extra- and intracellularpreservation of DNA. Gene expression is also enabled or increased bypreventing endosome acidification with NH₄Cl or chloroquine.Polyethylenimine, which facilitates gene expression without additionaltreatments, probably disrupts endosomal function itself. Disruption ofendosomal function has also been accomplished by linking to thepolycation endosomal-disruptive agents such as fusion peptides oradenoviruses.

[0038] Polycations can also facilitate DNA condensation. The volumewhich one DNA molecule occupies in a complex with polycations isdrastically lower than the volume of a free DNA molecule. The size of aDNA/polymer complex is probably critical for gene delivery in vivo. Interms of intravenous injection, DNA needs to cross the endothelialbarrier and reach the parenchymal cells of interest. The largestendothelia fenestrae (holes in the endothelial barrier) occur in theliver and have an average diameter of 100 nm. The trans-epithelial poresin other organs are much smaller, for example, muscle endothelium can bedescribed as a structure which has a large number of small pores with aradius of 4 nm, and a very low number of large pores with a radius of20-30 nm. The size of the DNA complexes is also important for thecellular uptake process. After binding to the cells the DNA-polycationcomplex should be taken up by endocytosis. Since the endocytic vesicleshave a homogenous internal diameter of about 100 nm in hepatocytes andare of similar size in other cell types, DNA complexes smaller than 100nm are preferred.

[0039] Condensation of DNA

[0040] A significant number of multivalent cations with widely differentmolecular structures have been shown to induce condensation of DNA.

[0041] Two approaches for compacting (used herein as an equivalent tothe term condensing) DNA:

[0042] 1. Multivalent cations with a charge of three or higher have beenshown to condense DNA. These include spermidine, spermine, Co(NH₃)₆³⁺,Fe³⁺, and natural or synthetic polymers such as histone H1,protamine, polylysine, and polyethylenimine. Analysis has shown DNAcondensation to be favored when 90% or more of the charges along thesugar-phosphate backbone are neutralized.

[0043] 2. Polymers (neutral or anionic) which can increase repulsionbetween DNA and its surroundings have been shown to compact DNA. Mostsignificantly, spontaneous DNA self-assembly and aggregation processhave been shown to result from the confinement of large amounts of DNA,due to excluded volume effect.

[0044] Depending upon the concentration of DNA, condensation leads tothree main types of structures:

[0045] 1) In extremely dilute solution (about 1 ug/mL or below), longDNA molecules can undergo a monomolecular collapse and form structuresdescribed as toroid.

[0046] 2) In very dilute solution (about 10 ug/mL) microaggregates formwith short or long molecules and remain in suspension. Toroids, rods andsmall aggregates can be seen in such solution.

[0047] 3) In dilute solution (about 1 mg/mL) large aggregates are formedthat sediment readily.

[0048] Toroids have been considered an attractive form for gene deliverybecause they have the smallest size. While the size of DNA toroidsproduced within single preparations has been shown to vary considerably,toroid size is unaffected by the length of DNA being condensed. DNAmolecules from 400 bp to genomic length produce toroids similar in size.Therefore one toroid can include from one to several DNA molecules. Thekinetics of DNA collapse by polycations that resulted in toroids is veryslow. For example DNA condensation by Co(NH₃)₆Cl₃ needs 2 hours at roomtemperature.

[0049] The mechanism of DNA condensation is not clear. The electrostaticforce between unperturbed helices arises primarily from a counterionfluctuation mechanism requiring multivalent cations and plays a majorrole in DNA condensation. The hydration forces predominate overelectrostatic forces when the DNA helices approach closer then a fewwater diameters. In a case of DNA-polymeric polycation interactions, DNAcondensation is a more complicated process than the case of lowmolecular weight polycations. Different polycationic proteins cangenerate toroid and rod formation with different size DNA at a ratio ofpositive to negative charge of 0.4. T4 DNA complexes with polyarginineor histone can form two types of structures; an elongated structure witha long axis length of about 350 nm (like free DNA) and dense sphericalparticles. Both forms exist simultaneously in the same solution. Thereason for the co-existence of the two forms can be explained as anuneven distribution of the polycation chains among the DNA molecules.The uneven distribution generates two thermodynamically favorableconformations.

[0050] The electrophoretic mobility of DNA-polycation complexes canchange from negative to positive in excess of polycation. It is likelythat large polycations don't completely align along DNA but form polymerloops that interact with other DNA molecules. The rapid aggregation andstrong intermolecular forces between different DNA molecules may preventthe slow adjustment between helices needed to form tightly packedorderly particles.

[0051] Preparation of polycation-condensed DNA particles is ofparticular importance for gene therapy, more specifically, particledelivery such as the design of non-viral gene transfer vectors. Optimaltransfection activity in vitro and in vivo can require an excess ofpolycation molecules. However, the presence of a large excess ofpolycations may be toxic to cells and tissues. Moreover, thenon-specific binding of cationic particles to all cells forestallscellular targeting. Positive charge also has an adverse influence onbiodistribution of the complexes in vivo.

[0052] Several modifications of DNA-cation particles have been createdto circumvent the nonspecific interactions of the DNA-cation particleand the toxicity of cationic particles. Examples of these modificationsinclude attachment of steric stabilizers, e.g. polyethylene glycol,which inhibit nonspecific interactions between the cation and biologicalpolyanions. Another example is recharging the DNA particle by theadditions of polyanions which interact with the cationic particlethereby lowering its surface charge, i.e. recharging of the DNAparticle. We have demonstrated that layering of polyelectrolytes can beachieved on the surface of DNA/polycation particles (V S Trubetskoy, ALoomis, J E Hagstrom, V G Budker, J A Wolff. Nucleic Acids Res.27:3090-3095, 1999, incorporated herein by reference). Another exampleis cross-linking the polymers and thereby caging the complex (V STrubetskoy, A Loomis, P M Slattum, J E Hagstrom, V G Budker, J A Wolff.Bioconjugate Chem. 10:624-628, 1999, incorporated herein by reference).Nucleic acid particles can be formed by the formation of chemical bondsand template polymerization. Trubetskoy et al used two types ofpolymerization reactions to achieve DNA condensation: steppolymerization and chain polymerization (V S Trubetskoy, V G Budker, L JHanson, P M Slattum, J A Wolff, L E Hagstrom. Nucleic Acids Res.26:4178-4185, 1998) U.S. patent application Ser. No. 08/778,657incorporated herein by reference.

[0053] The Use of pH-Sensitive Lipids, Amphipathic Compounds, andLiposomes for Drug and Nucleic Acid Delivery

[0054] After the landmark description of DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)[Felgner, P L, Gadek, T R, Holm, M, et al. Lipofection: a highlyefficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad.Sci. USA. 1987;84:7413-7417], a plethora of cationic lipids have beensynthesized. Basically, all the cationic lipids are amphipathiccompounds that contain a hydrophobic domain, a spacer, andpositively-charged amine. The hydrophilic domains are typicallyhydrocarbon chains such as fatty acids derived from oleic or myristicacid. The hydrocarbon chains are often joined either by ether or esterbonds to a spacer such as glycerol. Quaternary amines often compose thecationic groups. Usually, the cationic lipids are mixed with a fusogeniclipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to formliposomes. The mixtures are mixed in chloroform that is then dried.Water is added to the dried lipid film and unilamellar liposomes formduring sonication. Multilamellar cationic liposomes and cationicliposomes/DNA complexes prepared by the reverse-phase evaporation methodhave also been used for transfection. Cationic liposomes have also beenprepared by an ethanol injection technique.

[0055] Several cationic lipids contain a spermine group for binding toDNA. DOSPA, the cationic lipid within the LipofectAMINE formulation(Life Technologies) contains a spermine linked via a amide bond andethyl group to a trimethyl, quaternary amine [Hawley-Nelson, P,Ciccarone, V and Jessee, J. Lipofectamine reagent: A new, higherefficiency polycationic liposome transfection reagent. Focus 1993;15:73-79]. A French group has synthesized a series of cationic lipidssuch as DOGS (dioctadecylglycinespermine) that contain spermine [Remy,J-S, Sirlin, C, Vierling, P, et al. Gene transfer with a series oflipophilic DNA-binding molecules. Bioconjugate Chem. 1994;5:647-654].DNA has also been transfected by lipophilic polylysines which containdipalmotoylsuccinylglycerol chemically-bonded to low molecular weight(˜3000 MW) polylysine [Zhou, X, Kilbanov, A and Huang, L. Lipophilicpolylysines mediate efficient DNA transfection in mammalian cells.Biochim. Biophys. Acta 1991;1065:8-14. Zhou, X and Huang, L. DNAtransfection mediated by cationic liposomes containing lipopolylysine:Characterization and mechanism of action. Biochim. Biophys. Acta 1994;1195-203].

[0056] Other studies have used adjuvants with the cationic liposomes.Transfection efficiency into Cos cells was increased when amphiphilicpeptides derived from influenza virus hemagglutinin were added toDOTMA/DOPE liposomes [Kamata, H, Yagisawa, H, Takahashi, S, et al.Amphiphilic peptides enhance the efficiency of liposome-mediated DNAtransfection. Nucleic Acids Res. 1994;22:536-537]. Cationic lipids havebeen combined with galactose ligands for targeting to the hepatocyteasialoglycoprotein receptor [Remy, J-S, Kichler, A, Mordvinov, V, et al.Targeted gene transfer into hepatoma cells with lipopolyamine-condensedDNA particles presenting galactose ligands: A stage toward artificialviruses. Proc. Natl. Acad. Sci. USA 1995;92:1744-1748]. Thiol-reactivephospholipids have also been incorporated into cationic lipid/pDNAcomplexes to enable cellular binding even when the net charge of thecomplex is not positive [Kichler, A, Remy, J-S, Boussif, 0, et al.Efficient gene delivery with neutral complexes of lipospermine andthiol-reactive phospholipids. Biochem. Biophys. Res. Comm.1995;209:444-450]. DNA-dependent template process convertedthiol-containing detergent possessing high critical micelleconcentration into dimeric lipid-like molecule with apparently low watersolubility (J P Behr and colleagues).

[0057] Cationic liposomes may deliver DNA either directly across theplasma membrane or via the endosome compartment. Regardless of its exactentry point, much of the DNA within cationic liposomes does accumulatein the endosome compartment. Several approaches have been investigatedto prevent loss of the foreign DNA in the endosomal compartment byprotecting it from hydrolytic digestion within the endosomes or enablingits escape from endosomes into the cytoplasm. They include the use ofacidotropic (lysomotrophic), weak amines such as chloroquine thatpresumably prevent DNA degradation by inhibiting endosomal acidification[Legendre, J. & Szoka, F. Delivery of plasmid DNA into mammalian celllines using pH-sensitive liposomes: Comparison with cationic liposomes.Pharmaceut. Res. 9, 1235-1242 (1992)]. Viral fusion peptides or wholevirus have been included to disrupt endosomes or promote fusion ofliposomes with endosomes, and facilitate release of DNA into thecytoplasm [Kamata, H., Yagisawa, H., Takahashi, S. & Hirata, H.Amphiphilic peptides enhance the efficiency of liposome-mediated DNAtransfection. Nucleic Acids Res. 22, 536-537 (1994). Wagner, E., Curiel,D. & Cotten, M. Delivery of drugs, proteins and genes into cells usingtransferrin as a ligand for receptor-mediated endocytosis. Advance DrugDelivery Reviews 14, 113-135 (1994)].

[0058] Knowledge of lipid phases and membrane fusion has been used todesign potentially more versatile liposomes that exploit the endosomalacidification to promote fusion with endosomal membranes. Such anapproach is best exemplified by anionic, ph-sensitive liposomes thathave been designed to destabilize or fuse with the endosome membrane atacidic pH [Duzgunes, N., Straubinger, R. M., Baldwin, P. A. &Papahadjopoulos, D. PH-sensitive liposomes. (eds Wilschub, J. &Hoekstra, D.) p. 713-730 (Marcel Dekrer INC, 1991)]. All of the anionic,pH-sensitive liposomes have utilized phosphatidylethanolamine (PE)bilayers that are stabilized at non-acidic pH by the addition of lipidswhich contain a carboxylic acid group. Liposomes containing only PE areprone to the inverted hexagonal phase (H_(II)). In pH-sensitive, anionicliposomes, the carboxylic acid's negative charge increases the size ofthe lipid head group at pH greater than the carboxylic acid's pK andthereby stabilizes the phosphatidylethanolamine bilayer. At acidic pHwithin endosomes, the uncharged or reduced charge species is unable tostabilize the phosphatidylethanolamine-rich bilayer. Anionic,pH-sensitive liposomes have delivered a variety of membrane-impermeantcompounds including DNA. However, the negative charge of thesepH-sensitive liposomes prevents them from efficiently taking up DNA andinteracting with cells; thus decreasing their utility for transfection.We have described the use of cationic, pH-sensitive liposomes to mediatethe efficient transfer of DNA into a variety of cells in culture.

[0059] The Use of pH-Sensitive Polymers for Drug and Nucleic AcidDelivery

[0060] pH-sensitive polymers have found broad application in the area ofdrug delivery exploiting various physiological and intracellular pHgradients for the purpose of controlled release of drugs (both lowmolecular weight and polymeric). pH sensitivity can be broadly definedas any change in polymer's physico-chemical properties over certainrange of pH. More narrow definition demands significant changes inpolymer's ability to retain (release) bioactive substance (drug) inphysiologically tolerated pH range (usually pH 5.5-8). pH-sensitivitypresumes the presence of ionizable groups in the polymer (polyion). Allpolyions can be divided into three categories based on their ability todonate or accept protons in aqueous solutions: polyacids, polybases andpolyampholytes. Use of pH-sensitive polyacids in drug deliveryapplications usually relies on their ability to become soluble with thepH increase (acid/salt conversion), to form complex with other polymersover change of pH or undergo significant change inhydrophobicity/hydrophilicity balance. Combinations of all three abovefactors are also possible.

[0061] Copolymers of polymethacrylic acid (Eudragit S, Rohm America) areknown as polymers which are insoluble at lower pH but readilysolubilized at higher pH, so they are used as enteric coatings designedto dissolve at higher intestinal pH (Z Hu et al. J. Drug Target., 7,223, 1999). Typical example of pH-dependent complexation is copolymersof polyacrylate(graft)ethyleneglycol which can be formulated intovarious pH-sensitive hydrogels which exhibit pH-dependent swelling anddrug release (F Madsen et al., Biomaterials, 20, 1701, 1999).Hydrophobically-modified N-isopropylacrylamide-methacrylic acidcopolymer can render regular egg PC liposomes pH-sensitive bypH-dependent interaction of grafted aliphatic chains with lipid bilayer(O Meyer et al., FEBS Lett., 421, 61, 1998). Polymers with pH-mediatedhydrophobicity (like polyethylacrylic acid) can be used as endosomaldisruptors for cytoplasmic drug delivery (N Murthy et al. J. ControlledRelease 61, 137, 1999).

[0062] Polybases have found broad applications as agents for nucleicacid delivery in transfection/gene therapy applications due to the factthey are readily interact with polyacids. Typical example ispolyethyleneimine (PEI). This polymer secures nucleic acid electrostaticadsorption on the cell surface followed by endocytosis of the wholecomplex. Cytoplasmic release of the nucleic acid occures presumably viaso called “proton sponge” effect according to which pH-sensitivity ofPEI is responsible for endosome rupture due to osmotic swelling duringits acidification (O Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297,1995). Cationic acrylates possess the similar activity (for example,poly-((2-dimethylamino)ethyl methacrylate) (P van de Wetering et al. J.Controlled Release 64, 193, 2000). However, polybases due to theirpolycationic nature pH-sensitive polybases have not find broad in vivoapplication so far. They exhibit acute systemic toxicity in vivopresumably mostly of colloid nature (JH Senior, Biochim. Biophys. Acta,1070, 173, 1991). Milder polybases (for example, linear PEI) are bettertolerated and can be used systemically for in vivo gene transfer (DGoula et al. Gene Therapy 5, 712, 1998).

[0063] Blood Interactions of Delivery Complexes

[0064] Blood interactions are of importance for in vivo delivery ofdrugs and genes. Retroviral vectors are inhibited in human blood throughcomplement (C) activation (Russell, et al., Human Gene Therapy,6:635-641. 1995, Welsh, et al., Nature, 257:612-614. 1975, Rother, etal., Hum. Gene Ther., 6:429-435. 1995). Retroviral vectors produced innon-human cell lines contain galactosyl(α1-3)galactosyl(αGal) terminalsugars within glycolipids and glycoproteins (Cosset, et al., J. Virol.,69:7430-7436. 1995). Humans lack this sugar structure and produceantibodies against it (sensitized presumably by gut flora). Retroviralvectors produced in αGal-negative cell lines do not suffer this problem.Similar findings have been made for VSV, HIV-2, human foamy viruses andthe vectors derived from them (Takeuchi, et al., J. Virol.,71:6174-6178. 1997). Another mechanism for retroviral inactivationinvolves C activation via direct binding of C1q to the p 15 (envelope)transmembrane protein (Welsh, et al., Nature, 257:612-614. 1975,Pensiero, et al., Human Gene Therapy, 7:1095-1101. 1996, Bartholomew andEsser, Biochemistry, 19:2847-2853. 1980). Another viral vector system,baculovirus vectors (which can transduce mammalian hepatocytes), isinhibited by serum but not by C inactive serum (heat treated or depletedin C3 or C4) (Sandig, et al., Human Gene Therapy, 7:1937-1945. 1996).Other factors such as chondroitin sulfates within pleural effusionsinhibit retroviral vector gene transfer (Batra, et al., J. Biol. Chem.,272:11736-11741. 1997).

[0065] Of relevance to the development of non-viral vectors, is the vastliterature on the interactions of the C system and other serum factorswith liposomes (Szebeni, Crit. Rev Therap. Drug Carrier Systems,15:57-88. 1998). Opsonization of liposomes by serum proteins plays animportant role in their clearance by the reticuloendothelial system(RES). For example, clearance of negatively-charged liposomes is aidedby β2-glycoprotein I binding (Chonn, et al., J. Biol. Chem.,270:25845-25849. 1995). Generally, neutral liposomes are poor Cactivators but they are cleared similarly to anionic liposomes in vivo,possibly because they absorb anionic serum proteins in vivo (Devine andBradley, Advanced Drug Delivery Reviews, 32:19-29. 1998). “Plain”liposomes (phospholipid/cholesterol bilayers without antigeniccomponents) bind several serum proteins including albumin, IgG,extracellular matrix proteins (fibronectin, laminin, serum amyloidprotein), clotting factors, apolipoproteins, β2-glycoprotein-1, Creactive protein (CRP), α2-macroglobulin, and C factor such as C1q andC3 (Senior, Crit. Rev. Ther. Drug Carrier Syst., 3:123. 1987, Scherphof,et al., Liposome Technology, 205. 1984, Juliano, Liposomes, 53. 1983).Apo E binds to both anionic and neutral liposomes, but only the small,neutral liposomes had reduced liver targeting in apo E-deficient mice(Scherphof and Kamps, Advanced Drug Delivery Reviews, 32:81-97. 1998).This suggests that apo E-binding is important for the liver targeting ofneutral but not anionic liposomes. Anionic liposomes activate C via theclassical pathway starting with C1q (without anti-phosholipidantibodies), which leads to deposition of C3b and iC3b on the liposome'ssurface (Liu, et al., Biochim. Biophys. Acta, 1235:140-146. 1995)(Devine and Bradley, Advanced Drug Delivery Reviews, 32:19-29. 1998).Cationic liposomes activate C via the alternative pathway in human serumbut weakly in rat serum. Another pathway is due to CRP binding ofphosphocholine and galactosyl residues within liposomes (Volanakis andNarkates, J. Immunology, 126:1820-1825. 1981). The clearance ofliposomes is affected by several other factors such as their size,fluidity, packing, and cholesterol content. In summary, the clearance ofconventional liposomes is delayed by using small, neutral, unilamellarliposomes containing rigid bilayers (disteraroyl phosphatidylcholine orsphingomylein and cholesterol) (Lasic and Martin, Pharmacology andToxicology: Basic and Clinical Aspects, 1995). Besides affectingdelivery, C activation can also release anaphylatoxins (e.g., C3a, C4a,and C5a) that activate mast cells, basophils and platelets causingrespiratory, blood pressure and dermatologic sequelae.

[0066] Many of this field's concepts and experimental methods are nowbeing extended to the use of cationic lipids for DNA transfer. Seruminhibits the transfection with the use of several types of cationiclipids by modifying the DNA/cationic lipid complexes (Escriou, et al.,Biochim. Biophys. Acta, 1368:276-288. 1998, Zelphati and Szoka,Pharmaceutical Research, 13:1367-1372. 1996, Senior, et al., Biochimicaet Biophysica Acta, 1070:173-9. 1991, Felgner, et al., Proc. Natl. Acad.Sci. USA, 84:7413-7417. 1987, Zelphati, et al., Biochim. Biophys. Acta,1390:119-133. 1998, Li, et al., Gene Therapy, 5:930-937. 1998) (Yang andHuang, Gene Therapy, 5:380-387. 1998, Yang and Huang, Gene Therapy,4:950-960. 1997). Cationic lipid/DNA complexes activate C but it doesnot affect in vivo gene transfer (Barron, et al., Human Gene Therapy,9:315-323. 1998). DNA/cationic lipid complexes made with GL-67 did notactivate C because the complexes contain neutral lipids and have chargeclose to neutrality (Scheule, et al., Human Gene Therapy, 8:689-707.1997, Plank, et al., Human Gene Therapy, 7:1437-1446. 1996).

[0067] Prevention of Unfavorable Interactions

[0068] On the basis of the above paradigm, we could borrow from thearsenal of cellular and viral approaches for preventing bloodinactivation. For C mediated pathways, a variety of membrane (DAF, MIR1,CR1, MCP) and fluid-phase (C1 inh, C4bp, factor I, factor H) proteinsprevent C activation on native cells (Devine and Bradley, Advanced DrugDelivery Reviews, 32:19-29. 1998). Viruses borrow these C inhibitoryfactors from cells as a “cloak” to prevent C activation. For example,HIV type I uses decay-accelerating factor (CD55) to inhibit C activation(Marschang, et al., Eur. J. Immunol., 25:285-290. 1995). Vectors couldincorporate new chimeric or modified C regulatory proteins that arebeing developed to inhibit C activation (e.g., solubilized C3 convertaseinhibitor, modified CD55)(Ryan, Nature Medicine, 1:967-968.1995).

[0069] A “Dysopsonin” hypothesis has been proposed in which serumproteins can bind to foreign particles and prolong their bloodcirculation (Moghimi, et al., Biochim. Blophys. Acta, 1179:157-165.1993). For example, Moghimi and colleagues have reported that two serumproteins prevented the uptake of poloxamine (a block co-polymer ofpolyoxyethylene and polyoxypropylene)-coated microspheres by isolatedliver sinusoidal cells but did not identify the proteins. Ourpreliminary studies indicate that C-reactive protein (CRP) binding to T7phage can prolong their blood circulation by preventing phageinactivation by C. This work also suggests a new approach for targetingin which natural serum proteins selectively adhere to the deliveryparticle and provide it with targeting properties.

[0070] In terms of artificial delivery systems, incorporation ofspecific glycoplipids such as GMI ganglioside, cerebroside sulfate, orphosphatidylinositol or PEG (polyethylene glycol) prolongs thecirculation time of liposomes in the blood by providing “stericstabilization”(Lasic and Martin, Pharmacology and Toxicology: Basic andClinical Aspects, 1995). PEG and other hydrophilic polymers have beenincorporated into a variety of polycation- and cationic lipid-containinggene transfer systems (Eastman, et al., Human Gene Therapy, 8:765-73.1997, Toncheva, et al., Biochim. Biophys. Acta, 1380:354-368. 1998,Wolfert, et al., Human Gene Therapy, 7:2123-33. 1996, Astafieva, et al.,FEBS Lett., 389:278-80. 1996, Meyer, et al., J. Biol. Chem.,273:15621-7. 1998, Katayose and Kataoka, J. Pharm. Sci., 87:160-163.1998, Maruyama, et al., Bioconj. Chem., 8:735-742. 1997, Ferdous, etal., J. Pharm. Sci., 87:1400-1404. 1998, Ferdous, et al., Nucleic AcidRes., 26:3949-3954. 1998, Asayama, et al., Bioconj. Chem., 9:476-481.1998, Vinogradov, et al., Bioconj. Chem., 9:805-812. 1998, Choi, et al.,Bioconjugate Chem., 9:708-718. 1998). Despite the promise of PEG, itsuse can be challenging. For example, it's attachment can interfere withcell surface receptor interactions and endocytosis (Lasic and Martin,Pharmacology and Toxicology: Basic and Clinical Aspects, 1995, Lasic,1997). PEGylation of adenoviral vectors also interferes withtransduction. Sialic acid-containing molecules such as glycophorin (amajor RBC sialoglycoprotein), GM₃ (a principal sialoglycolipid), GM1 andheparin have been incorporated into liposomes in order to mimic themembrane molecules on cells that inhibit C activation (Okada, et al.,Immunology, 48:129. 1983, Okada, et al., J. Immunol., 134:3316. 1985,Michalek, et al., J. Immunol., 140:1581. 1988, Michalek, et al., J.Immunol., 140:1588. 1988, Shichijo and Alving, Biochim. Biophys. Acta.,858:118. 1986). The inhibitory effect of these compounds was abrogatedby removal of sialic acid with neuraminidase digestion. Sialic acidsurfaces preferentially bind factor H which inhibits the alternativepathway by preventing factor B binding to C3b.

[0071] Phage Display Systems a Powerful Approach for Enhancing GeneDelivery:

[0072] The idea of using peptide ligands for targeting drug and genedelivery vehicles (Cotten, M. & Wagner, E. Receptor-mediated genedelivery strategies. (eds Friedmann, T.) p. 261-279 (Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1999)., Reynolds, P. N. & Curiel,D. T. Strategies to adapt adenoviral vectors for gene therapyapplications: Targeting and integration. (eds Friedmann, T.) p. 111-130(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1999)) and forconstructing biocompatible materials (Healy, et al., Ann. N.Y. Acad.Sci., 875:24-35. 1999, Shakesheff, et al., Journal of BiomaterialsScience, Polymer Edition, 9:507-18. 1998) is widely accepted due to itsconceptual and technical simplicity. A number of peptides with tissuetargeting properties were selected both in vitro and in vivo by usingthe peptide libraries displayed at the amino-terminus of the filamentousphage coat proteins pIII or pVIII (Healy, et al., Ann. N.Y. Acad. Sci.,875:24-35. 1999, Shakesheff, et al., Journal of Biomaterials Science,Polymer Edition, 9:507-18. 1998, Pasqualini and Ruoslahti, Nature,380:364-6. 1996, Pasqualini, et al., Nat. Biotechnol., 15:542-546. 1997,Rajotte, et al., J. Clin. Invest., 102:430-437. 1998, Rajotte andRuoslahti, J. Biol. Chem., 274:11593-8. 1999, Samoylova and Smith,Muscle Nerve, 22:460-6. 1999, Pasqualini, Quart. J. Nucl. Med.,43:159-62. 1999, Koivunen et al., Meth. Mol. Biol. 129:3-17. 1999,Koivunen et al., Nature Biotech., 17:768-74. 1999, Kassner et al.,Biochem. Biophys. Res. Comm., 264:921-8. 1999, Koivunen et al., J. Nucl.Med., 40:883-8. 1999, Ivanenkov et al., Biochim. Biophys. Acta,1448:463-72. 1999, Ivanenkov et al., Biochim. Biophys. Acta,1448:450-62. 1999, Barinaga, Science, 279:323-4. 1998, Arap et al.,279:377-80. 1998, Folkman, Nature Biotech. 15:510).

[0073] Libraries of small peptides have been used to map epitopes,protein-protein interactions, protease inhibitors, integrin ligands, andreceptor agonists and antagonists (Cwirla, et al., Proc. Natl. Acad.Sci. USA, 87:6378-6382. 1990, Scott and Smith, Science, 249:386-390.1990, O'Neil et al., Proteins, 14:509-15. 1992, Smith, et al., J. Biol.Chem., 270:6440-9. 1995, Doorbar and Winter, J. Mol. Biol., 244:361-9.1994, Hong and Boulanger, EMBO J., 14:4714-27.1995, Ferrer and Harisson,J. Virology, 73:5795-802. 1999, Kola et al., Mol. Immunol., 36:145-52.1999). Phages displaying larger polypeptides such as antibodies,hormones, enzymes, and DNA-binding proteins have also been screened(Lowman, et al., Biochem., 30:10832-8. 1991, Rebar and Pabo, Science,263:671-3. 1994, Soumillion, et al., J. Molec. Biol., 237:415-22. 1994,Roberts, et al., Proc. Natl. Acad. Sci. USA, 89:2429-33. 1992).

[0074] Of note is the work that is using phage display libraries todevelop gene transfer methods (Russell, Nature Med., 2:276-277. 1996).Peptides that bind to shared receptors on different cell lines have beenobtained by alternating rounds of biopanning among the different cells(Goodson, et al., Proc. Natl. Acad. Sci. USA, 91:7129-33. 1994).Following injection of a peptide library into the tail vein of mice,brain and kidney-specific peptide sequences were identified and used forspecific in vivo targeting of red blood cells (Pasqualini and Ruoslahti,Nature, 380:364-366. 1996). A phage containing the integrin-binding RGDpeptide was internalized by cultured human laryngeal epithelial cells(Hart, et al., J. Biol. Chem., 269:12468-12474. 1994). Usingbacteriophage, a peptide that binds α₉β₁-integrin was identified forincorporation into non-viral vectors that are able to transfect airwayepithelia (Schneider, et al., FASEB Letters, 429:269-273. 1998). Anothergroup screened an M13 (pIII, 20-mer) library and selected for phage thatcan be internalized by cells in culture and that had affinity for musclecells (Barry, et al., Nature Med., 2:299-305. 1996). Recently,bacteriophage libraries were used to identify peptides that can targetGST-fusion proteins to lung endothelium (Rajotte, et al., J. Clin.Invest., 102:430-437. 1998). Interestingly, a pIII/M13 27-mer peptidedisplay phage library was used to identify a C3-binding peptide (bypanning for C3b-binding phages) that inhibits human C but not rat ormouse C (Sahu, et al., J. Immunol., 157:884-891. 1996).

[0075] Of relevance is a study that selected intraperitoneally injectedλ bacteriophage for long circulation in the blood (Merril, et al., Proc.Natl. Acad. Sci. USA, 93:3188-3192. 1996). Two bacteriophage clones wereselected. One had a glutamic acid to lysine substitution in position 158(not the carboxy terminus) of the major λ capsid head protein E. Anotherclone had this same mutation plus an uncharacterized mutation in the λcapsid head protein D. The mechanism by which these mutations enabledprolonged circulation was not characterized.

[0076] Methods of Producing Antibodies

[0077] The techniques currently used in production of antibodies fallinto several groups. The oldest approach uses serum from immunizedanimals as a source of antibodies The antigen can be injected indifferent forms, at different locations and at different times intoanimals of different species. The resultant serum can be used as is orantibodies with a different degree of purity can be isolated from serumusing various precipitation, extraction, chromatographic andelectrophoretic techniques or their combinations (Harlow and Lane.Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988).

[0078] Antibodies can be also produced in vitro, using the hybridomatechnology. by isolating B-cells from the animals pre-immunized with aparticular antigen and growing them in culture. The isolated cells areimmortalized by fusing them with myeloma cells that do not produceimmunoglobulins of their own. The resultant hybrids are cloned and theclones that secret antibodies against the antigen of interest areselected and propagated further either in culture or as ascites. Thesecreted antibodies are monoclonal antibodies (Yokoyama WM. Productionof monoclonal antibodies. (Eds. Coligan et al.,) Current Protocols inImmunology. New York: John Willey and Sons, 1995:2.5.1-2.5.17, Harlowand Lane. Antibodies: a laboratory manual. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor. 1988,).

[0079] An alternative apprach to hybridoma technology is based on theuse of gene libraries and expression systems. This allows to avoidlabor-intensive immunizations of animals and screening of supernatants.Besides, this approach allows to circumvent tolerance. V_(H) and V_(L)libraries are prepared separately and then combined into a combinatoriallibrary by cleaving, mixing and religating the libraries at arestriction site (Huse et al., Science, 246:1275-1281. 1989, Clackson etal., Nature, 352:624-28. 1991). V_(H) and V_(L) can both be expressed onone covalent polypeptide (Clackson et al., Nature, 352:624-28. 1991).

[0080] The power of the combinatorial approach is considerably enhancedby using phage display libraries, where V_(H) and V_(L) genes areexpressed on the surface of phage particles as fusion protein with thephage coat protein. This approach permits screening of a large number ofsequences (Clackson et al., Nature, 352:624-28. 1991, McCafferty et al.,Nature, 348:552-554. 1990). The selection can be started with as many as10¹⁰ clones prepared from “naive” B-cells. Selected combinations ofV_(H) and V_(L) genes can be recombined into “hierachic libraries” andselection repeated (McCafferty et al., Nature, 348:552-554. 1990).

[0081] The increase in the affinity can also be achieved by mutatingselected clones using such techniques as growing phage in the mutD5 E.coli strain with an error-prone DNA-polymerase III, “shuffling” ofselected sequences, error-prone PCR and site-directed mutagenesis (Lowet al., J. Mol. Biol., 260:359-368. 1996, Thompson et al., J. Mol.Biol., 256:77-88. 1996).

[0082] Another important development in production of monoclonalantibodies was designing antibodies with some or all structure derivedfrom human immunoglobulins. Such antibodies have lower immunogenicity inhumans and, therefore, higher therapeutic potential. Several approacheshave been taken based of fusion of human cells with animal myelomas orwith human tumor cells or immortalization of human cells by usingEpstein-Barr virus (Cole et al., Proc. Natl. Acad. Sci., 80:2026-2030.1983, Olsson and Kaplan, Methods Enzymol., 92:3-16. 1983, Seigneurin etal., Science, 221:173-175). The recombination of selected animalvariable regions with human constant regions gives so-called chimericantibodies (Morrison S L. Science, 229:1202-1207. 1985, Morrison et al.,Proc. Natl. Acad. Sci. 81:6851-6855. 1984). The additional substitutionof animal framework sequences in the variable regions by human onesgives so called “humanized” antibodies (Jones et al., Nature,321:522-525. 1986). Some additional engineering work is typicallyrequired to optimize the exposure of selected portions of the animalvariable region in the new scaffold. Completely human antibodies can beproduced now in transgenic mice (Lonberg et al., Nature, 368:856-859.1994, Green et al., Nat. Genet., 7:13-21. 1994).

[0083] Single-stranded antibodies can be produced in a soluble form inE. coli. The incorporation of specific, “dimerizing” sequences intosingle-stranded antibodies with different specificities allows one toproduce bispecific antibodies composed of two connected antibodymolecules. One of the most powerful uses of such molecules isredirecting cytolytic cells to defined targets (Karpovsky et al., J.Exp. Med., 160:1686-1701. 1984, Titus et al., J. Immunol.,138:4018-4022. 1987). Another interesting application of bi-specificantibodies is changing the viral tropism (Wickham et al., J. Virol.,70:6831-6838).

[0084] Uses of Antibodies for Therapeutic Purposes A number of in vivoapplications for monoclonal antibodies have been developed (Waldmann TA, Science, 252:1657-62. 1991, Berkower I., Curr. Opin. Biothechnol.,7:622-8. 1988). Leukemias and lymphomas have so far been the favoritetargets for in vivo therapy based on the use of monoclonal antibodies(Appelbaum F R., Sem. Hemat., 36(4 Suppl. 6):2-8. 1999, Bendandi andLongo, Curr. Opin. Oncol., 11:343-50. 1999). Numerous attempts to treatother types of cancer have been undertaken as well (Reviewed byWawrzyczak E J, Antibody therapy, Oxford, UK:Bios Scientific Publishers.1995, Weiner L M., Sem. Oncol., 26 (4 Suppl. 14):43-51. 1999, Sem.Oncol. (4 Suppl. 12):41-50). The antibodies can be used either bythemselves, relying on the antibody effector functions, or as conjugateswith various toxins and radionuclides (Weiner L M., Sem. Oncol., 26 (4Suppl. 14):43-51. 1999, Sem. Oncol. (4 Suppl. 12):41-50). Antibodieshave been also used for a variety of other applications, such astargeting platelets (Gensini et al., Am. Heart J. (2 Pt. 2), 138:171-6.1999), blocking T-lymphocytes in rejection reactions (Ortho MulticenterTransplant Study Group, N. Engl. J. Med., 313:337-342), intercepting LPSfor treatment of sepsis, blocking IL-6 receptor for treatment ofmultiple myeloma, and membrane IgE for treatment of allergy (Reviewed byBerkower I., Curr. Opin. Biothechnol., 7:622-8. 1988).

[0085] Uses of Antibodies for Diagnostic Purposes Most currentapplications of antibodies serve diagnostic rather than curativepurposes. In vitro, they are widely used in RIA and ELISA measurementsof substances in biological fluids, from hormones to toxins. They arealso indispensable in flow cytometric assays. In vitro, the antigens tobe identified are typically fractionated prior to the reaction withantibodies and immobilized on special supports. The antigen-antibodycomplexes are visualized by using secondary antibodies conjugated withfluorescent or electron-scattering labels or enzymes that digestspecific substrate and cause the location precipitation of resultantproducts. In vivo, antibodies are used as tumor-imaging reagents(Collier et al., Radiology, 185:179-186, 1992).

SUMMARY

[0086] The high complexity of available peptide display libraries(10⁷-10⁹) should allow the selection of peptides with almost anytargeting specificity. Our invention indicates, however, that thebehavior of displayed peptides in vivo is more complex than currentlyrealized. We show that immobilized epitopes such as peptides with freecarboxy-termini are specifically recognized by natural antibodies andinduce rapid activation of complement (C). Recognition of displayedpeptides by natural antibodies has not previously been reported. Severalpossibilities flow from this discovery which include: I) Process ofselection for phage that are less prone to inactivation, II) Productionof peptide-display phage libraries that are less prone to inactivation,III) Phage that are less prone to inactivation for treating bacterial,IV) Methods for selection of serum proteins that bind specific peptideligands, V) Peptides for drug and nucleic acid delivery, and VI) Methodsfor the production and uses of peptide-specific natural antibodies(PSNA).

[0087] The selection pressure against T7 phage with displayed peptidesin blood provides an unique opportunity for selecting peptides thatprotect the phage against C by binding to blood proteins. In rat blood,new peptide ligands were identified that protected the phage againstC-mediated inactivation by binding C-reactive protein (CRP). In humanserum, a number of peptides with tyrosine residues preserved phageinfectivity, presumably by binding human α₂-macroglobulin.

[0088] We have found that an excess of UV-irradiated phage will protectthe live phage with the same peptides on the surface against seruminactivation in vitro and in vivo. However, the UV-irradiated phage willnot protect live phage with the peptides that have very differentstructures. In other words, serum inactivation of a specific T7 phageclone is inhibited by an excess amount of UV-irradiated phage from thesame class of clones but not from other classes. This indicates that thepeptides in the phage library belong to structurally and functionallydifferent classes that activate complement through the interaction withdifferent peptide-binding macromolecules. The differences betweendistinct classes of peptides may be defined by such parameters as size,shape, charge and hydrophobicity. This also suggests that serum containsa class of molecules that recognizes a specific class of peptides andactivates complement (C). Given that the serum was from naive animals orpooled human serum, it could not simply be due to antibodies that arisefrom immune activation. Use of serum depleted of IgM indicates that itis at least peptide-specific IgM species that recognize the T7 phageclones and activate complement (C). Natural antibodies have beenwell-described but they are supposed to be “polyreactive” withouthigh-affinity peptide specificity. Thus, it appears that mammals have aninnate system for recognizing particles (or surfaces) coated withmultiple copies of specific peptides.

[0089] Peptide-display phage libraries that are less prone toinactivation can be produced using this knowledge of the inactivationmechanism and the phage (and their associated proteins and peptides)that are resistant to inactivation. For example, a “double-display”phage library can be made so that each phage has two different proteinsin its coat; “protein A” that affects its interactions with blood ortissue and “protein B” that contains a “random” peptide sequence.Protein A is derived from the phage clones that are selected forpersistence in circulation in animals and for resistance to inactivationin serum in vitro or in vivo.

[0090] This recognition process based upon peptide-specific naturalantibodies (PSNA) and complement activation may also play a role innormal and pathologic processes. For example, protease digestion ofviruses, cell surfaces or internal structures (e.g. myofibers, myelin orjoint surfaces) may lead to the display of several carboxy terminatedpeptides that are likely to be recognized by peptide-specific IgM. Thiscould be beneficial in the clearance of viruses but destructive intissues such as joints or myelin.

[0091] The discovery of peptide-specific natural antibodies (PSNA) hasseveral important applications. PSNA's can be purified from the sera ofhumans or animals or be produced recombinantly. These PSNA's can be usedfor diagnostic purposes for detecting a variety of disorders in humansand other living organisms (animals and plants and microogranisms) thatinclude infectious disease, auto-immune disorders and disorders with anautoimmune component (such as rheumatoid arthritis, systemic lupuserythrematosus (SLE), multiple schlerosis, ankylosing spondylitis,psoriasis, Reiter's Syndrome, fibromylagia, dermatomyositis,polymyositis, schleroderma, diabetes mellitius, glomerulonephritis), andcancer. These PSNA's could also be used to treat human and animaldisorders that include cancer, infectious disease, auto-immune disordersand aspects of other disorders.

BRIEF DESCRIPTION OF THE DRAWING

[0092]FIG. 1. The percentage of phage recovered (phage recovered/phageinjected X 100, mean of 2 experiments) of various T7 phage clones fromthe blood and liver 5 min after tail vein injections in mice in vivo.Our laboratory names for the T7 phage corresponding to their C-terminussequences are shown below. T7 vector refers to the vector codingsequence without any library inserts.

[0093] Methods: The phages (109 pfu/0.3 ml PBS) were injected into tailvein of ICR male mice (5-6 weeks old) pre-treated (20-24 h) with GdCl3at a dose of 10 mg/kg. Three min after injection, heparin (20 unit/head)was injected by same route. Under anesthesia, blood was collecteddirectly from heart and livers were perfused with 1 unit heparin/mlcontaining PBS 30 ml for 3 min, and then collected. Livers werehomogenated with 3-fold of their weight of 2% Triton X-100/1M NaCl inPBS to lyse the cells and disperse the phage. The titration of phageswas assessed by using E. coli BL21 in an appropriate dilution. 20/6 FQ*32/77 FQS* 32/23 FSQV* #112 FQSGVMLGDPN* #114 . . .FQSGVMLGDPNSDGALRQSGRGKSSRP* T7 Vector FQSGVMLGDPNSSSVDKLAAALE*

DETAILED DESCRIPTION

[0094] The invention is described in the following sections: I) Methodsof selection for phage that are less prone to inactivation, II)Peptide-display libraries and production of peptide-display phagelibraries that are less prone to inactivation, III) Phage that are lessprone to inactivation for treating bacterial infections, IV) Methods forselection of serum proteins that bind specific peptide ligands, V)Peptides for drug and nucleic acid delivery, infections, VI) Methods forthe production and uses of peptide-specific natural antibodies (PSNA).

[0095] I. Methods of Selection for Phage that are Less Prone toInactivation

[0096] We have discovered that bacteriophage (abbreviated as “phage”)are inactivated in blood in vivo (e.g., in the systemic circulation ofan animal) and in serum in vitro (in a test tube outside of the body) bynatural antibodies and complement. The test tube indicates any type ofcontainer for holding a liquid and can be made of glass or plastic.Inactivation means the loss of the phage's ability to infect bacteria.Phage infection can be assessed based on the ability of phage to lysebacterial lawns. Therefore, the amount of phage is expressed in plaqueforming units that correspond to the number of plaques (small clearareas) in a lawn of bacteria grown on an agar plate.

[0097] The phage can be inactivated by antibodies that activatecomplement upon binding to the phage. The antibodies can be naturalantibodies that mostly belong to the IgM or IgG class. The phage canalso be inactivated if it is biotinylated prior to exposure to thebodily fluid (e.g., blood in vivo or in vitro) and then allowed tointeract with a probe that tightly binds biotin, such as avidin,streptavidin, neutravidin, or other protein derived from or related tothese proteins.

[0098] In one embodiment of the invention, this inactivation process isused to select for phage that are resistant to the inactivation process.The initial phage for this selection process can be (but not limited to)peptide display phage libraries that are derived from filamentous phagesuch as M13 or non-filamentous phage such as T7 phage. The initial phagecan also be UV-irradiated to increase the number of mutations or derivedfrom recombinant DNA that has been subjected to regular polymerase chainreaction (PCR), error-prone PCR, or DNA shuffling or method to increasethe variability or number of mutations in the phage sequence.

[0099] For the in vitro testing, blood or a blood derived material suchas plasma, serum, or purified proteins and factors can be used in theinactivation process. For the in vitro testing, serum derived from blood(by clotting) or citrated plasma (by adding Ca) can be used in theinactivation process. The complement-grade serum from commercial sourcesthat is pre-filtered and lyophilized or snap-frozen can also be used.Prior to use, the serum pH can be left as is or adjusted to 7.2-7.6. Incase of commercial serum, it is also filtered to remove proteinprecipitate formed during freezing or lyophilization. With the serumdepleted of particular proteins, purified proteins such asimmunoglobulins (Ig) as a whole or Ig fractions that are enriched forIgG, IgM, IgA, or IgD can be used to reconstitute the ability of serumto inactivate phage. Purified factors that may be required for phageinactivation or protection by incomplete serum also include complementproteins, blood fractions containing proteins greater than 25 kDa(kilodaltons) 50 kDa, 100 kDa, 200 kDa or 300 kDa in size. Blood, serum,plasma, or other blood-derived fractions can be subjected topurification processes that include precipitation, extraction, columnchromatography methods and electrophoresis. Column chromatographymethods include the following types of chromatography: size exclusion,ion-exchange, reverse phase, affinity purification or any combination ofthese types. Electrophoresis includes the types of separation based oncharge, pI, the change of protein mobility in the presence of particularligands or any combination of these techniques.

[0100] For in vivo testing in the whole animal, the phage can beinjected intravenously or intraarterially into the systemic or pulmonarycirculation. It can also be injected into blood vessels that supply theliver (hepatic artery, portal vein, hepatic vein, via the vena cava, viathe aorta), kidneys, muscle (femoral, iliac, brachial, axilla arteries)or brain. The phage can also be injected into other body spaces thatinclude the peritoneum (intraperitoneally) and cerebral spinal fluid(ventricular spaces, subdural, epidural). The animal can be a vertebratesuch as fish, amphibians, reptiles or mammals. Mammals include rodents(mice, rats), guinea pigs, hamsters, dogs, pigs, non-human primates(such as Rhesus) and humans. The animals can be particular strains withcertain features. These can be animals that are defective in the immunesystem and do not produce certain antibodies, for instance,IgM-deficient mice. These animals can also have defects in thecomplement system, such as the absence or functional impairment ofparticular complement proteins caused by natural or artificial deletionsor mutations in the corresponding genes.

[0101] Phage that are resistant to inactivation can be obtained from avariety of tissues in vivo, including the blood, liver, lung, brain,muscle, spleen, kidneys, intestines, prostate, thymus, adrenal glands,thyroid, gonads, eyes, and skin. The resistance to inactivation resultsfrom association of phage with particular plasma proteins that protectphage against inactivation by the complement system. Different plasmaproteins bind to phage through specific recognition of differentpeptides or proteins or protein domains displayed at the phage surface.Such complexes of phage with plasma proteins can either stay incirculation or be targeted to particular locations through theinteraction of bound plasma proteins with cell receptors. The binding ofbound plasma proteins to cell receptors is promoted by changes in theconformation of bound proteins and by a high level of cooperation on theprotein-receptor interactions due to a high density of bound proteins onthe phage surface. Therefore, the phage binds to particular organsthrough indirect targeting. Alternatively, if the peptides are exposedon the phage in the format that is not recognized by natural antibodies,the phage can be used to select for the peptides that bind to cellreceptors directly.

[0102] In one embodiment to measure the survival of phage T7 in blood,10⁹ pfu of phage per animal are injected into a tail vein and 100 μlblood samples are collected from a non-injected tail vein (of the sameanimal) into 10 μl (10U) of heparin on ice at specified time points.Plasma is prepared by centrifugation. Phage from blood and plasma aredetected by soft agar plating. Plating dilutions are done in LB medium(20 g of yeast extract, 40 g of trypton and 20 g of NaCl per 1 L ofmedium).

[0103] In a preferred embodiment, phage are subjected to more than oneround of selection. Phage that are selected for resistance toinactivation are then grown on bacteria to expand their number and thensubjected to another inactivation process. This can change the type ofphage clones that are resistant to inactivation. It can also increasethe percentage of phage that survive the inactivation process.

[0104] In other embodiments, factors that affect the inactivationprocess can be added to the test tube in vitro or to the living animal.These include small molecules or drugs such as phosphoryl choline andaminocaproic acid. Molecules that inhibit macrophage activity includegadolinium (GdCl₃), carrageenan and incapsulated bisphosphonates.Molecules that inhibit the complement system include antibodies, venoms(e.g cobra venom factor—CVF) and natural or artificial complementregulatory proteins represented by both membrane and soluble proteins.The function of the complement system in vitro can be also inhibited byheat (50° C. for the alternative complement activation pathway and 56°C. for the classical complement activation pathway) and chelators (EGTAor EDTA for the alternative complement activation pathway and EDTA orEGTA or EGTA/Mg for the classical complement activation pathway).

[0105] II. Peptide-Display Libraries and Production of Peptide-DisplayPhage Libraries That are Less Prone to Inactivation

[0106] T7-based peptide display libraries are made by using Novagen T7vectors. Both the vectors that give the phage with a high number ofpeptide copies per phage particle (displayed in all coat proteins) andthe vectors that give just 0.1-10 copies of peptides of polypeptides perphage particles are used. Low-copy phage are grown either in the E. colistrains provided by Novagen (BLT5403 and BLT5615) or other strains. Thebulk of the phage coat protein comes from the plasmid but the structureof this protein is different. The E. coli strains BLT5403 and BLT5615produce T7 protein 10A. In other libraries, the bulk of the phage coatprotein is a truncated 10B protein that shows a remarkable protection ofphage against complement-mediated inactivation.

[0107] “Double” display libraries displaying: a) a “constant” peptide orprotein that prevents serum inactivation (e.g. lys+/arg+ peptides) or isless prone to inactivation and b) a random peptide for selection oftissue, sub-cellular, or blood persistence properties.

[0108] For making “double” display libraries of T7 phage, bacteria suchas E. coli are modified so that it expresses a T7 phage coat proteinthat is incorporated into phage that constitute a peptide-displaylibrary. It is double-display in that each phage has two differentproteins in its coat; “protein A” that affects its interactions withblood or tissue and “protein B” that contains a “random” peptidesequence. This peptide sequence is “random” in that it is differentamong different phage clones that constitute a phage library. In oneembodiment, protein A is selected for resistance to serum inactivationin vitro or prolonged blood circulation in vivo. In one embodimentprotein A is derived from the phage clone 20-6 (Table 3) (AAGAVVFQpeptide sequence for coat protein carboxy-terminus).

[0109] Selected Sequence:MASMTGGQQMGTNQGKGVVAAGDKLALFLKVFGGEVLTAFARTSVTTSRHMVRSISSGKSAQFPVLGRTQAAYLAPGENLDDKRKDIKHTEKVITIDGLLTADVLIYDIEDAMNHYDVRSEYTSQLGESLAMAADGAVLAEIAGLCNVESKYNENIEGLGTATVIETTQNKAALTDQVALGKEIIAALTKARAALTKNYVPAADRVFYCDPDSYSAILAALMPNAANYAALIDPEKGSIRNVMGFEVVEVPHLTAGGAGTAREGTTGQKHVFPANKGEGNVKVAKDNVIGLFMHRSAVGTVKLRDLALERARRANFQADQIIAKYAMGHGGLRPEAAGAVVFQ

[0110] In other embodiments, protein A is derived from the phage clone32-77 (AAGAVVFQS peptide sequence for coat protein carboxy-terminus) orphage clone 32-23 (AAGAVVFSQV peptide sequence for coat proteincarboxy-terminus) (Table 3). In yet other embodiments, protein A isderived from phage clones listed in Table 1 and have a terminal lysine(Table 1A) or arginine (Table 1B) or contain a tyrosine (Table 1E). Thegene encoding these different “protein A” are placed within a plasmidthat is carried by a bacteria such as E. coli.

[0111] “Protein B” contains a “random” peptide sequence. This peptidesequence is “random” in that it is different among different phageclones that constitute a phage library. The random peptide sequence canbe at the amino terminus, carboxy terminus or within the coat protein.The random peptide sequence can encode a peptide that is one to 25 aminoacid residues in length. The random peptide can contain invariant partsin addition to the random part. In one embodiment, the invariant part isderived from phage clones listed in Table 1 and has a terminal lysine(Table 1A) or arginine (Table 1B) or contains a tyrosine (Table 1E).

[0112] T7 phage libraries constructed with proteins “A” and “B” can havevarying proportions of these proteins. In one embodiment, protein Aconstitutes 0.1, 1, 10, 20, or 50 percent of the coat proteins in thephage. Proteins A and B can contain two cys residues so that disulfidebonds are formed and peptide sequences are constrained.

[0113] In one embodiment, T7 libraries displaying random peptides withinthe 10B capsid protein are constructed using a random one- to 25-merpeptide insert by using simple second strand synthesis (O'Neil, et al.,Methods in Enzymology, 245:370-86. 1994) and placed into the Eco RI/HindIII sites of the T7Select 415-1 vector arms (Novagen Corp., Madison,Wis.). The single-stranded sequence is an oligonucleotide(xxxGAATTCggacggtgcc (NNG/T)1-25 ggggctggaAAGCTTxxxxxx). A 21-merreverse primer (xxxxxxAAGCTTtccagcccc) is used to fill in thecomplementary strand with exo⁻ Klenow fragment. Specific methods forcloning, propagation and maintenance are used as specified in the manualsupplied with the T7Select Kit (Novagen). The complexity of ourlibraries generated by growing phage in the BL21 E. coli strain isdetermined.

[0114] In order to be able to draw on long peptides, along with shortones, two other E. coli host strains, BLT5615 and BLT5403 in addition toBL21m can be used. Both strains provide additional phage T7 coatprotein, 10A, from a plasmid. According to our observations, the ratioof 10A and 10B proteins in the phage based on the vector T7Select415-1is 4 to 1. A lower density of long peptides on the phage surfacepromotes phage survival in a mixed population. Decreasing the density ofdisplayed peptides should be also useful while trying to select forhigh-affinity peptide ligands. It enables one to determine the number ofpeptides required for a certain effect.

[0115] Constrained phage T7 display containing X₂CX₃₋₂₀CX₂ peptides canalso be used. It may be necessary to expose the phage to gentleoxidizing agents to form the disulfide bonds. It has been found thatconstrained peptide display libraries may be more apt for finding aspecific ligand but this may not be necessary for large peptides thatcan form secondary structure.

[0116] III. Phage for Treating Bacterial Infections

[0117] Peptide-display phage that are less prone to inactivation can beused to treat bacterial infections. In one embodiment, a phage libraryis selected for clones that are resistant to serum inactivation in vitroand the clones resistant to serum inactivation are injected into ananimal with a bacterial infectious disorder. The phage infects thebacteria and kills the bacteria, thus alleviating the infectious diseasestate in the animal. The phage can be injected intravenously or into thetissue that is infected such as sinuses, pulmonary, prostate,gastrointestinal, or central nervous system (ventricular fluid, brainparenchyma, spinal cord). In one embodiment, the phage is a T7 phage. Inanother embodiment, the T7 phage is the phage clone 20-6 (Table 3)(AAGAVVFQ peptide sequence for coat protein carboxy-terminus).

[0118] IV. Selection of Serum Proteins that Bind Specific PeptideLigands

[0119] We have discovered that phage are rapidly inactivated by blood,serum and other blood derivatives and factors in vitro and in thecirculating blood in vivo. Phage clones can be selected that areresistant to this inactivation process. In some instances, the phageclones that are resistant to inactivation bind to blood constituentssuch as serum proteins. In a preferred embodiment a blood constituentbinds to the displayed peptide sequence that is unique or specific tothat particular phage clone. (That is, each phage clone displays aspecific peptide sequence that is related to a DNA sequence and that isspecific to the specific phage clone. Depending on the complexity of thephage library the phage clone and thus the peptide sequence may or maynot be unique.)

[0120] The blood constituent that binds to the peptide (cognate to thephage clone that is resistant to inactivation) can be purified andidentified on the basis of the affinity of the blood constituent for thepeptide. In one embodiment, affinity purification can be accomplished byaffinity chromatography.

[0121] Yet in another embodiment, affinity purification can be done byaffinity centrifugation. In another embodiment, affinity purificationcan be performed using affinity precipitation. For example, antibodiesagainst the phage can be used to immunoprecipitate the phage with theblood constituent attached to the phage. In another embodiment, affinitypurification can be brought about by filtering. In one instance, filtersthat do not let the phage pass through filters can be used to purify thephage and the blood constituent attached to it away from the rest of theblood constituents that do not bind the phage as tightly.

[0122] In all these types of affinity purification, the phage can bewashed to enrich for a blood constituent that binds the phage clone withdifferent affinities or via different methods. The washes can containvarious concentrations of salt. The washes can also contain detergents.The washes can also contain specific ligands such as phosphatidylcholine, free peptides, and peptides attached to proteins and othersupports.

[0123] In a preferred embodiment, after the blood constituent and thephage are purified from the rest of the blood, the blood constituentbound to the phage is separated from the phage and the blood constituentis identified using techniques such as protein gel electrophoresis,two-dimensional electrophoresis, immunoblotting, and protein sequencing.

[0124] V. Peptides for Biologically-Active Compound and Nucleic AcidDelivery

[0125] Phage clones that are resistant to inactivation can be used toderive peptides that are of use for drug and nucleic acid delivery. Inone embodiment, they are peptides that have a terminal lysine orarginine at the carboxy terminus. In another embodiment, they arepeptides that contain a tyrosine. In yet another embodiment, they arepeptides that have a terminal lysine or arginine at the carboxy terminusor they are peptides that contain a tyrosine and that are used toprolong the circulation life or persistence of the biologically-activecompound or nucleic acid delivery particle in the circulation or renderthe drug or nucleic acid delivery particle more resistant toinactivation.

[0126] In still another embodiment, the peptide contains a celltargeting signal and a terminal lysine or arginine at the carboxyterminus. The peptide can also contain a cell targeting signal and atyrosine. The peptides that contain a cell targeting signal and aterminal lysine or arginine at the carboxy terminus and that are used toprolong the circulation life or persistence of the biologically-activecompound or nucleic acid delivery particle in the circulation or renderthe drug or nucleic acid delivery particle more resistant toinactivation.

[0127] A biologically-active compound or nucleic acid delivery particleconsists of a biologically-active compound or nucleic acid andamphipathic compounds. In another embodiment, the drug or nucleic aciddelivery particle can consist of a drug or nucleic acid and liposomes.Also, the drug or nucleic acid delivery particle can consist of a drugor nucleic acid and a polymer. The polymer can be a polyion such as apolycation or polyanion. Yet in another embodiment, the drug or nucleicacid delivery particle can consist of a drug or nucleic acid, anamphipathic compound, and a polymer. Still, in another embodiment, thepeptide could have the carboxy terminus blocked so that there is notunmodified carboxy group at the “carboxy” terminus or the peptide couldhave the amino terminus blocked so that there is no unmodified aminogroup at the “amino” terminus.

[0128] In yet another embodiment, random peptides or the peptides withthe primary structure corresponding to the primary structure of theproteins from the recipient species are used to target nucleic acids ordrug carriers to the liver. Also, the delivery particles derivatizedwith random peptides or the peptides with the structure derived from theproteins of the recipient species are used to decrease immunogenicity ofconjugated peptides.

[0129] VI. Peptide-Specific Natural Antibodies

[0130] Peptide-specific natural antibodies (PSNA) are natural antibodiesthat reacts specifically with a peptide in some demonstrable way such asinducing inactivation of phage that display multiple peptides. It canalso be demonstrated by enzyme linked immunoassay (ELISA) orradioimmunoassay (RIA) or by affinity chromatography.

[0131] In one embodiment, the PSNA's are IgM or IgG.

[0132] In another embodiment, PSNA's are purified from serum or plasmaby using synthetic peptides conjugated to the resin. The PSNA's bound tothe peptides on the column can be eluted by low pH or high pH buffers,by the buffers containing chaotropic agents or high salt or by thebuffers that combine any of these properties. The PSNA's with lowaffinity can be isolated by isocratic elution of the column with thesame buffer that was used to apply serum or plasma on to the column.

[0133] In another embodiment, PSNA's are produced using recombinantmethods-include phage as well. V_(H) and V_(L) immunoglobulin genes areisolated from a pool of “naive” B-cells and used to constructsingle-chain antibodies exposed either on M13 or T7 phage. The phage ispanned against UV-inactivated, immobilized T7 phage displaying specificpeptides or a whole library. Alternatively, the phage is panned againstimmobilized synthetic peptides. The bound live phage is collected andamplified. The selection process is repeated and selectedimmunoglobulins characterized in terms of their affinity toward targetpeptides and the ability to activate C if converted into a physiologicalform recognized by C1q.

[0134] In yet another embodiment, PSNA's can be produced usingmonoclonal methods. B-cells producing PSNA's are be selected by usingfluorescently labeled phage displaying peptides of interest. Theselected B-cells are immortalized using conventional techniques, clonedand the antibodies secreted by resultant clones are characterized fortheir ability to bind peptides of interest and activate C once bound.

[0135] In another embodiment, PSNA-producing B-cells are selected byusing antiidiotypic antibodies that specifically bind to PSNA's.

[0136] PSNA's can be used for diagnostic or detection purposes. Theseinclude detecting the presence of a particular peptide sequence in aprotein. The protein containing the specific peptide sequence or apeptide can be in a mixture such as in an extract from a tissue or atissue section. The protein or peptide can be on a membrane, glass, orplastic structure. The protein or tissue section could be first digestedwith an enzyme and then probed with the PSNA. Binding of the PSNA to thepeptide sequence can be detected by a variety of methods. These includethe attachment of a reporter or marker molecule directly to the PSNA orindirectly. In one embodiment, another antibody or secondary antibodycontaining a reporter or marker molecule is used to detect the presenceof the PSNA. The PSNA can be used in immunoblots, ELISA, RIA,immunohistochemical assays, fluorescence polarization, or Biocore-typebinding assays.

[0137] PSNA's can also be used for selective visualization or labelingof particular tissues or cells. PSNA's can be indirectly selected forthis purpose from a natural pool of PSNA's or from B-cell libraries byusing the phage that displays a repertoire of peptides characteristic ofcertain cells or tissues. Such phage can be selected by pre-incubatingserum with the cells or tissues of interest followed by collecting thephage that survives in the serum pre-incubated with the target tissue.Prior to these steps, the target cells can be pre-treated with proteasesthat would generate exposed protein carboxy-termini or permeabilizedwith detergents or fixed and then permeabilized with detergents. Thetreatment of cells with detergent is used to expose proteincarboxy-termini on the inner side of the cell plasma membrane and on thecell organells. A combination of proteolysis and permeabilization canalso be used. The pre-existing and/or protease-generated repertoire ofcarboxy-termini on the plasma membrane and cell organelles can bedifferent for different types of cells. Therefore, different PSNA'swould be removed from the serum pre-exposed to different cells. Thephage displaying corresponding peptides would survive in the absence ofcell- or tissue-specific PSNA's and could be used after a few rounds ofselection as a probe for the isolation of B-cells producing PSNA'sagainst these peptides. Corresponding synthetic peptides could be usedfor affinity purification of pre-existing PSNA's that would selectivelyreact with different cells and tissues. The staining of cells or tissueswith selected PSNA's can be done using conventional secondary antibodiesor by using the antibodies against C proteins. In the latter case, thecomplement deposition will be specifically induced by the multivalentbinding of PSNA's to closely spaced protein carboxy-termini. Spuriousbinding events will not generate complement activation and, therefore,will not be detected.

[0138] A similar approach can be used to generate PSNA's that wouldstain particular protein bands or spots immobilized on paper, plastic orglass supports. The immobilized proteins could be incubated with serumas is or after mild treatment with proteases. The serum depleted ofparticular PSNA's would be used to select phage with correspondingpeptides.

[0139] The different individual types of PSNA's can be correlated withthe prevalence, incidence, penetration, or likelihood of an individualhaving a specific disease or disorder. This would be akin to correlatinga particular genotype to a phenotype. In fact, the genetic loci forencoding the PSNA's could be used for this purpose as well.

[0140] PSNA's can also be used to treat disease in living creatures andorganisms such as animals and humans. The disease can be, but notlimited to, an infectious disease, cancer, autoimmune disorder,inflammatory condition, cardiovascular disorder, or nervous disorder.The PSNA can be injected into the blood or a tissue. It can be a part ofthe PSNA such as the part the binds the peptide. The PSNA that is usedfor therapeutic purposes can be purified from a bodily fluid, producedby recombinant methods, or by “monoclonal” methods. The PSNA can be“humanized” in that parts of the PSNA that are non-essential for bindingto the peptide are removed and replaced with parts of human antibodies.The PSNA can also be linked to another biologically-active compound orprotein such as a toxin (e.g., diptheria or pertusis)

[0141] Definitions

[0142] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0143] Biologically Active Compound

[0144] A biologically-active compound is a compound having the potentialto react with biological components. More particularly, biologicallyactive compounds utilized in this specification are designed to changethe natural processes associated with a living cell. For purposes ofthis specification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. In this specification, the cellular production of, orinhibition of a material, such as a protein, caused by a human assistinga molecule to an in vivo cell is an example of a delivered biologicallyactive compound. Pharmaceuticals, proteins, peptides, polypeptides,hormones, cytokines, antigens, viruses, oligonucleotides, enzymes andnucleic acids are examples of biologically active compounds.

[0145] Peptide and polypeptide refer to a series of amino acid residues,more than two, connected to one another by amide bonds between thealpha-amino group and carboxyl group of contiguous amino acid residues.The amino acids may be naturally occurring or synthetic. Polypeptideincludes proteins and peptides, modified proteins and peptides, andnon-natural proteins and peptides. Enzymes are proteins evolved by thecells of living organisms for the specific function of catalyzingchemical reactions. A chemical reaction is defined as the formation orcleavage of covalent or ionic bonds. Bioactive compounds may be usedinterchangeably with biologically active compound for purposes of thisapplication.

[0146] Delivery of Biologically-Active Compound

[0147] The delivery of a biologically-active compound is commonly knownas “drug delivery”. “Delivered” means that the biologically-activecompound becomes associated with the cell or organism. The compound canbe in the circulatory system, intravessel, extracellular, on themembrane of the cell or inside the cytoplasm, nucleus, or otherorganelle of the cell.

[0148] Parenteral routes of administration include intravascular(intravenous, intraarterial), intramuscular, intraparenchymal,intradermal, subdermal, subcutaneous, intratumor, intraperitoneal,intrathecal, subdural, epidural, and intralymphatic injections that usea syringe and a needle or catheter. An intravascular route ofadministration enables a polymer or polynucleotide to be delivered tocells more evenly distributed and more efficiently expressed than directinjections. Intravascular herein means within a tubular structure calleda vessel that is connected to a tissue or organ within the body. Withinthe cavity of the tubular structure, a bodily fluid flows to or from thebody part. Examples of bodily fluid include blood, cerebrospinal fluid(CSF), lymphatic fluid, or bile. Examples of vessels include arteries,arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bileducts. The intravascular route includes delivery through the bloodvessels such as an artery or a vein. An administration route involvingthe mucosal membranes is meant to include nasal, bronchial, inhalationinto the lungs, or via the eyes. Other routes of administration includeintraparenchymal into tissues such as muscle (intramuscular), liver,brain, and kidney. Transdermal routes of administration have beeneffected by patches and ionotophoresis. Other epithelial routes includeoral, nasal, respiratory, and vaginal routes of administration.

[0149] Nucleic Acid

[0150] The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nuclei acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). The term encompasses sequences that include any of the knownbase analogs of DNA and RNA including, but not limited to,4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0151] DNA may be in the form of anti-sense, plasmid DNA, parts of aplasmid DNA, product of a polymerase chain reaction (PCR), vectors (P1,PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimericsequences, chromosomal DNA, or derivatives of these groups. RNA may bein the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (smallnuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-senseRNA, ribozymes, chimeric sequences, or derivatives of these groups.

[0152] “Anti-sense” is a polynucleotide that interferes with thefunction of DNA and/or RNA. This may result in suppression ofexpression. Natural nucleic acids have a phosphate backbone, artificialnucleic acids may contain other types of backbones and bases. Theseinclude PNAs (peptide nucleic acids), phosphothionates, and othervariants of the phosphate backbone of native nucleic acids. In addition,DNA and RNA may be single, double, triple, or quadruple stranded.

[0153] The term “recombinant DNA molecule” as used herein refers to aDNA molecule that is comprised of segments of DNA joined together bymeans of molecular biological techniques. “Expression cassette” refersto a natural or recombinantly produced polynucleotide molecule that iscapable of expressing protein(s). A DNA expression cassette typicallyincludes a promoter (allowing transcription initiation), and a sequenceencoding one or more proteins. Optionally, the expression cassette mayinclude trancriptional enhancers, non-coding sequences, splicingsignals, transcription termination signals, and polyadenylation signals.An RNA expression cassette typically includes a translation initiationcodon (allowing translation initiation), and a sequence encoding one ormore proteins. Optionally, the expression cassette may includetranslation termination signals, a polyadenosine sequence, internalribosome entry sites (IRES), and non-coding sequences.

[0154] A nucleic acid can be used to modify the genomic orextrachromosomal DNA sequences. This can be achieved by delivering anucleic acid that is expressed. Alternatively, the nucleic acid caneffect a change in the DNA or RNA sequence of the target cell. This canbe achieved by homologous recombination, gene conversion, or other yetto be described mechanisms.

[0155] Gene

[0156] The term “gene” refers to a nucleic acid (e.g., DNA) sequencethat comprises coding sequences necessary for the production of apolypeptide or precursor (e.g., —myosin heavy chain). The polypeptidecan be encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ non-translated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

[0157] As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0158] As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequencewhich encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

[0159] The term “isolated” when used in relation to a nucleic acid, asin “an isolated oligonucleotide” or “isolated polynucleotide” refers toa nucleic acid sequence that is identified and separated from at leastone contaminant nucleic acid with which it is ordinarily associated inits natural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

[0160] Gene Expression

[0161] As used herein, the term “gene expression” refers to the processof converting genetic information encoded in a gene into RNA (e.g.,mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e.,via the enzymatic action of an RNA polymerase), and for protein encodinggenes, into protein through “translation” of mRNA. Gene expression canbe regulated at many stages in the process. “Up-regulation” or“activation” refers to regulation that increases the production of geneexpression products (i.e., RNA or protein), while “down-regulation” or“repression” refers to regulation that decrease production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

[0162] Delivery of Nucleic Acids

[0163] The process of delivering a polynucleotide to a cell has beencommonly termed “transfection” or the process of “transfecting” and alsoit has been termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The delivery of nucleic acidcan lead to modification of the DNA sequence of the target cell.

[0164] The polynucleotides or genetic material being delivered aregenerally mixed with transfection reagents prior to delivery. The term“transfection” as used herein refers to the introduction of foreign DNAinto eukaryotic cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics.

[0165] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

[0166] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells whichhave taken up foreign DNA but have failed to integrate this DNA. Theterm “naked polynucleotides” indicates that the polynucleotides are notassociated with a transfection reagent or other delivery vehicle that isrequired for the polynucleotide to be delivered to a cell.

[0167] A “transfection reagent” or “delivery vehicle” is a compound orcompounds that bind(s) to or complex(es) with oligonucleotides,polynucleotides, or other desired compounds and mediates their entryinto cells. Examples of transfection reagents include, but are notlimited to, cationic liposomes and lipids, polyamines, calcium phosphateprecipitates, histone proteins, polyethylenimine, and polylysinecomplexes (polyethylenimine and polylysine are both toxic). Typically,when used for the delivery of nucleic acids, the transfection reagenthas a net positive charge that binds to the polynucleotide's negativecharge. For example, cationic liposomes or polylysine complexes have netpositive charges that enable them to bind to DNA or RNA.

[0168] Polymer

[0169] A polymer is a molecule built up by repetitive bonding togetherof smaller units called monomers. A polymer is defined as a compoundcontaining more than two monomers. A monomer is a compound that can beattached to itself or another monomer and thus a form a polymer.

[0170] In this application, the term polymer includes both oligomerswhich have two to about 80 monomers and polymers having more than 80monomers. The polymer can be linear, branched network, star, comb, orladder types of polymer. The polymer can be a homopolymer in which asingle monomer is used or can be copolymer in which two or more monomersare used. Types of copolymers include alternating, random, block andgraft.

[0171] Polyion

[0172] A polycation is a polymer containing a net positive charge, forexample poly-L-lysine hydrobromide. The polycation can contain monomerunits that are charge positive, charge neutral, or charge negative,however, the net charge of the polymer must be positive. A polycationalso can mean a non-polymeric molecule that contains two or morepositive charges. A polyanion is a polymer containing a net negativecharge. The polyanion can contain monomer units that are chargenegative, charge neutral, or charge positive, however, the net charge onthe polymer must be negative. A polyanion can also mean a non-polymericmolecule that contains two or more negative charges. The term polyionincludes polycation, polyanion, zwitterionic polymers, and neutralpolymers. The term zwitterionic refers to the product (salt) of thereaction between an acidic group and a basic group that are part of thesame molecule.

[0173] Cell Targeting Signals

[0174] Cell targeting signal (or abbreviated as the Signal) is definedin this specification as a molecule that modifies a biologically activecompounds such as drug or nucleic acid and can direct it to a celllocation (such as tissue) or location in a cell (such as the nucleus)either in culture or in a whole organism. By modifying the cellular ortissue location of the foreign gene, the function of thebiologically-active compound can be enhanced.

[0175] The cell targeting signal can be a protein, peptide, lipid,steroid, sugar, carbohydrate, (non-expresssing) polynucleic acid orsynthetic compound. The cell targeting signal enhances cellular bindingto receptors, cytoplasmic transport to the nucleus and nuclear entry orrelease from endosomes or other intracellular vesicles. The celltargeting signal can be a ligand that binds to its cognate receptor.

[0176] Nuclear localizing signals enhance the targeting of thepharmaceutical into proximity of the nucleus and/or its entry into thenucleus. Such nuclear transport signals can be a protein or a peptidesuch as the SV40 large T ag NLS or the nucleoplasmin NLS. These nuclearlocalizing signals interact with a variety of nuclear transport factorssuch as the NLS receptor (karyopherin alpha) which then interacts withkaryopherin beta. The nuclear transport proteins themselves could alsofunction as NLS's since they are targeted to the nuclear pore andnucleus. For example, karyopherin beta itself could target the DNA tothe nuclear pore complex. Several peptides have been derived from theSV40 T antigen. These include a short NLS (H-CGYGPKKKRKVGG-OH) or longNLS's (H-CKKKSSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH andH-CKKKWDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH). Other NLS peptides havebeen derived from M9 protein (CYNDFGNYNNQSSNFGPMKQGNFGGRSSGPY), E1A(H-CKRGPKRPRP—OH), nucleoplasmin (H-CKKAVKRPAATKKAGQAKKKKL-OH), andc-myc (H-CKKKGPAAKRVKLD-OH).

[0177] Signals that enhance release from intracellular compartments(releasing signals) can cause DNA release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin A1 and the ER-retaining signal (KDEL sequence),viral components such as influenza virus hemagglutinin subunit HA-2peptides and other types of amphipathic peptides.

[0178] Cellular receptor signals are any signal that enhances theassociation of the biologically active compound with a cell. This can beaccomplished by either increasing the binding of the compound to thecell surface and/or its association with an intracellular compartment,for example: ligands that enhance endocytosis by enhancing binding thecell surface. This includes agents that target to the asialoglycoproteinreceptor by using asiologlycoproteins or galactose residues. Otherproteins such as insulin, EGF, or transferrin can be used for targeting.Peptides that include the RGD sequence can be used to target many cells.Chemical groups that react with sulfhydryl or disulfide groups on cellscan also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as lipids fatty acids,cholesterol, dansyl compounds, and amphotericin derivatives. In additionviral proteins could be used to bind cells.

[0179] Amphipathic Compounds

[0180] Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties; carbohydrates,polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobicgroups.

[0181] Peptides

[0182] Peptides are polymers of amino acid residues and theirderivatives. The peptide contains 1 or more amino acids and can besynthesized by artificial synthetic methods or in a living organism. Theamino acid residues are joined by peptide bonds. A peptide bond is onewhich the carboxy group of one amino acid is united with the amino groupof another amino acid, with elimination of a molecular of water, thusforming a peptide bond: —CO—NH—. The peptide can contain amino acidderivatives or analogs such as d-forms of amino acids or β-amino acids.

[0183] Blood and Its Constituents

[0184] According to Stedman's dictionary, blood is the “fluid and itssuspended formed elements that circulated through the heart, arteries,capillaries, and veins. The constituents of the blood are thenon-cellular parts: serum, plasma, and the cellular parts: red bloodcells, white blood cells (leukocytes), and platelets. Plasma is thefluid (non-cellular) part of the blood of the blood that isdistinguished from the serum obtained after coagulation. Serum is thefluid portion of the blood after removal of the fibrin clot and bloodcells, distinguished from the plasma in circulating blood. Thenon-cellular parts of blood also consist of complement and clottingfactors. A blood product is a substance that is formed from blood orpurified from blood.

[0185] Complement (C)

[0186] It is a thermolabile substance, normally present in serum, thatis destructive to certain bacteria and other cells which have beensensitized by specific complement-fixing antibody. The C systemcomprises more than 30 plasma or membrane proteins. The activation of Crelies on a cascade of proteolytic steps performed by the proteasedomains in the components involved. There three distict C activationpathways: the classical pathway triggered by target-bound antibody, theMBLectin pathway triggered by polysaccharide structures of microbes, andthe alternative pathway triggered by the recognition of exogenousstructures by the C components themselves. For historical reasons, thecomponents of the C system are numbered from C1 to C9, with thebiochemical reaction sequence being C₁-C₄-C₂-C₃-C₅-C₆-C₇-C₈-C9.

[0187] C1 (a complex of three subunits, C1q, C1r, and C1s) afteractivation by antibody-antigen complex or other activators is enzymic(as C1 esterase) for C4 and (owning to the reaction with C4) for C2. TheC42 moiety (C3 convertase) of the C142 complex then cleaves C3, theactive fragment of which enters the C1423 complex that cleaves C5. Thecomplex C14235 then combines sequentially with C6, C7, C8, and C9 forform lytic complemnt. C1 may be activated also by aggregated antibody.C3 may also be activated by bacterial endotoxin, by the properdinsystem, and by a component of cobra venom.

[0188] Antibodies

[0189] One or other classes of globulins (immunoglobulins) present inthe blood serum or body fluids of an animal. The classes include IgG,IgM, IgA, IgD and IgE.

[0190] Natural Antibodies

[0191] Natural antibodies are antibodies whose appearance in the blooddoes not require immunization with the corresponding antigen. Accordingto Stedman's Medical Dictionary (Williams and Wilkins Co.,Baltimore—23^(rd) edition, 1976), “Originally, antibody was a body orsubstance evoked in man or other animal by an antigen, and characterizedby reacting specifically with the antigen in some demonstrableway—antibody and antigen each being defined in terms of the other, butit is now supposed that antibodies may also exist naturally withoutbeing present as a result of the stimulus provided by the introductionof an antigen.

[0192] Phage

[0193] Phage is also known as bacteriophage and is a virus that infectsbacteria or has an affinity for bacteria. They can contain either DNA orRNA. They may contain either single- or double-stranded nucleic acid.They can have various shapes and sizes. They can use different bacterialstrains or species as a host. They can lyze the infected cells or justuse the cells for reproduction.

[0194] Reporter or Marker Molecules

[0195] Reporter or marker molecules are compounds that can be easilydetected. Typically they are fluorescent compounds such as fluorescein,rhodamine, Texas red, cy 5, cy 3 or dansyl compounds. They can bemolecules that can be detected by infrared, ultraviolet or visiblespectroscopy or by antibody interactions or by electron spin resonance.Biotin is another reporter molecule that can be detected by labeledavidin. Biotin could also be used to attach targeting groups.

EXPERIMENTAL SECTION Example 1 Selection for Phage Persisting in Blood

[0196] We studied the interaction of displayed peptides with bloodconstituents using a T7 phage display library based on the NovagenT7Select415-1 vector. Each phage particle displayed 415 copies of acorresponding peptide at the carboxy-terminus of all copies of the phagecoat protein 10B (Novagen T7Select System Manual, 1996). The phage withdisplayed peptides mimicked fairly well the delivery vehicles with thepeptide ligands linked to the vehicles through the peptideamino-termini.

[0197] We found that the titer of the T7 phage peptide library in theplasma of rats preinjected with GdCl₃ (to inhibit macrophages (Mizgerd,et al., J. Leukoc. Biol., 59:189-95. 1996)) decreased by 95-99% within 5min after phage injection. The rapid decrease in the phage titer was notaccompanied by phage accumulation in blood cells, liver, kidneys,spleen, lungs, heart and skeletal muscles (data not shown). Therefore,the phage appeared to be functionally inactivated in blood. This wasconfirmed by observing phage inactivation in rat serum in vitro. Typicalrecovery of phage after incubating phage library (10⁷ pfu) with 100 μlof serum at 37° C. for 30 min was less than 1%.

[0198] In contrast to the phage library, wild-type T7 survived in vivoquite well. 15-20% of wild-type phage was recovered from plasma in 5 minafter injection. Since the library phage and wild-type phage weredifferent only in the structure of the carboxy-terminal part of theircoat proteins (Novagen T7Select System Manual, 1996), this suggestedthat the inactivation of the library was associated, at least in part,with the structure of the protein 10B C-termini displaying peptides.

[0199] The mechanism of phage inactivation by blood constituents wasstudied using non-selected phage and the phage selected for persistencein the blood of rats over 60 min in an infectious state. The phage yieldfrom the plasma of rats in the 1^(st) round of selection wasapproximately 0.01%. The yield rose to approximately 15% in the 2^(nd)round and stayed around this level through 3 following rounds ofselection. After the 5^(th) round, 33 individual clones were isolatedand sequenced. All sequenced clones displayed peptides with either a Kor an R residue at the carboxy-terminus (Table 1, A and B). These cloneswere hence designated K+ and R+ clones (K⁺/R+ as a class), respectively.Besides the carboxy-terminal K and R residues, there appeared to bepreference for certain amino acid residues at other peptide positions(marked in bold in Table 1, A and B). Non-selected phage were randomlyisolated from the initial library and divided into K⁺/R+ and K−/R−clones (Table 1, C and D). TABLE 1 Primary structure of selected andnon-selected T7 phage clones. Clone Peptide A. Selected K+ Phage 19-4QVTK 19-5 AK 19-6 VVVESVPK 19-7 ARPVQK 19-9 GRLK 19-15 AFTNK 19-16VTPQVK 19-17 AVK 19-18 DNTPKTK 19-19 SLK 19-20 HRPKEGGKPALK 19-22RTNPKVK 19-24 TTRTPK 19-25 NNAQGARVK 19-28 MATVK 19-29 KLRMK 19-30GVREPK 19-31 PTIK 19-32 SRASVKGSTK 19-33 IK 19-35 TK 19-37 KTK 19-38RKPQK 19-40 KVREK B. Selected R+ Phage 19-1 GGR 19-3 ASRVR 19-10 RER19-11 KSGGPAER 19-13 RRRNFER 19-14 MDSMSNTPNGSER 19-21 PSSQQAQR 19-36KNMR 19-39 QR C. Non-Selected K+/R+ Phage IL-2 AVK IL-7 QLVRVISR IL-15 RL5-3 NSR L5-7 RKSLR L5-10 RK D. Non-Selected K−/R− Phage IL-1 IEFSGIL-8 * IL-9 MVLPFQQTVA IL-11 QSANI IL-14 KIPY IL-16 LPSGG IL-20 YNAKTDRGIL-21 L IL-23 KTNVEKGPM IL-27 NSNAGLENH IL-32 IQL L5-1 ME L5-8 MVRRVL5-9 LSARAP E. Selected Y+ Phage 32-2 RSYR 32-3 QESRTETDSQYLA 32-5 QGDYT32-16 MQYS 32-17 YRA 32-22 YGPQQ 32-24 VDY 32-26 GKGKTDDPRYQKFT 32-28AATGSDQGLNKAY

[0200] Both selected and non-selected K+/R+ phage persisted in plasma ininfectious state much longer than K−/R− phage (FIG. 1A). None of thetested K−/R− clones, featuring altogether 10 different carboxy-terminalpeptide residues (Table 1D), showed significant presence in plasma (FIG.1A). Therefore, the persistence of K+/R+ phage in circulation was mainlydue to the presence of a K or an R residue at the peptidecarboxy-terminus. K+/R+ clones, in contrast to K−/R− clones, also showeda high level of survival in serum (Table 2, A, D and G). This suggesteda common mechanism for K+/R+ phage protection in vivo and in vitro.TABLE 2 The effect of various conditions on phage inactivation in vitro.Clone Serum Treatment Additions % Phage Survival* A. K+ none none 52.0 ±19.6 B. K+ Lysine-Sepharose none 1.8 ± 2.1 C. K+ Lysine-Sepharose CRP53.5 ± 19.8 D. R+ none none 44.5 ± 9.0  E. R+ Lysine-Sepharose none 0.4± 0.5 F. R+ Lysine-Sepharose CRP 37.0 ± 13.3 G. K−/R− none none 0 I.K−/R− Lysine-Sepharose none 0.3 ± 0.3 J. K−/R− Lysine-Sepharose CRP 0.1± 0.3 K. K+/R+ none 200 mM PC 0 #volume of the samples was adjusted to500 μl with PBS/0.68 mM CaCl₂. The samples were incubated at 37° C. for30 min, and examined for the presence of infectious phage by a plaqueforming assay (Novagen, Novagen T7 Select System Manual, 1996). The#phage survival percentage was calculated using the data for 5 differentclones with the same type of the protein 10B carboxy-terminus.]

[0201] Methods:

[0202] T7 Phage Peptide Display Library. The vector T7Select415-1

[0203] (NSSVDKLAAALE) (Novagen, Madison, Wis.) was employed to displayrandom peptides (NSDGA(X)₂₀ GAVKLAAALE) at the carboxy-terminus of allphage coat protein 10B molecules (Novagen T7Select System Manual, 1996).The expression of the second coat protein, 10A, was disabled (NovagenT7Select System Manual, 1996). The vector insert was generated from theoligonucleotide xxxGAATTCggacggtgcc (NNG/T)₂₀ ggggctggaAAGCTTxxxxxx,where N is any of the four nucleotides and xxx are the nucleotides addedto the 3′ and 5′ ends in order to enhance the efficiency of restrictiondigestion. The oligonucleotide was made double-stranded with Klenowfragment, using a reverse primer xxxxxxAAGCTTtccagcccc. After EcoR I andHind III restriction enzyme digestion, the insert was ligated intoT7Select415-1 vector (Novagen T7Select System Manual, 1996). The vectorwas packaged (Novagen T7Select System Manual, 1996) and an aliquot ofthe packaging mixture was used to estimate the complexity of the libraryby a plaque forming assay as described below. The apparent complexity ofthe library was 10⁸. Packaged phage was amplified in a log phase 0.5 Lculture of the BL21 E. coli strain at 37° C. for 4 hs. The cell debriswas removed by centrifugation and the phage was precipitated with 8%polyethylene glycol (M.W. 8,000) (Novagen T7Select System Manual, 1996).Phage was extracted from the pellet with 10 mM Tris-HCl/1M NaCl/1 mMEDTA (pH 8.0) and stored in PBS containing 10% glycerol at −80° C.(Novagen T7Select System Manual, 1996). The amplified library used inselection experiments displayed only truncated peptides, 1-14 amino acidresidues long, immediately at the 10B protein carboxy-terminus (Table1). The clones with full-size peptide inserts were lost duringamplification. UV-inactivated phage was prepared by irradiating 1 ml ofphage (10¹² pfu/ml; PBS) in a well of a 6-well tissue culture platepretreated with BSA for 15 min under constant stirring. A germicidallamp (15W; Sylvania G15T8) positioned 8 cm from the sample served as asource of UV.

[0204] In Vivo Studies. Long-circulating T7 phage was selected using180-200 g Sprague-Dawley female rats pre-injected with GdCl₃ (10 mg/kgbody weight) (Mizgerd, et al., J. Leukoc. Biol., 59:189-95. 1996) a daybefore. The animals were anesthetized with Ketamine (80 mg/kg) andXylazine (4 mg/kg) and 1010 pfu (plaque-forming units) of phage in PBSin the first or 10⁹ pfu in the following selection rounds were injectedinto a tail vein. After 60 min, 6 ml of blood were collected from twoanimals into 600 units of heparin (Elkins-Sinn, Inc.) and phage inplasma were amplified as above. Individual clones were analyzed by PCRcycle sequencing.

[0205] Persistence of phage in plasma was assessed by injecting 10⁹ pfuof phage into a tail vein. At specified time points, 100 μl bloodsamples were collected from a non-injected tail vein into 10 U ofheparin on ice. Phage titers in blood samples on ice did not diminishover time. After centrifugation at 14,000 rpm for 5 min and 1-3×10³ folddilution of the samples with LB medium, the amount of infectious phagewas determined using a plaque forming assay (Novagen T7Select SystemManual, 1996). Briefly, 10 μl of diluted plasma were incubated with 250μl of log phase BL21 E. coli cells for 5 min, mixed with 3 ml of 0.7%agar in LB medium and plated onto 1.5% agar in 10 cm plates. The plaqueswere allowed to develop overnight at room temperature or over 4 hs at37° C. The total amount of circulating phage was calculated based on a6.4 ml plasma volume for 200 g rats (Lee and Blaufox, J. Nucl. Med.,26:72-6. 1985). C activity was inhibited by pre-injecting ratsintraperitoneally with 100 μg/kg of CVF (Calbiochem) 20 hs before thephage injection. In PC co-injection experiments, phage was injected inPBS containing 10 mM PC (Na salt).

[0206] Serum preparation. Rat serum was prepared by clotting blood onice. Lysine-Sepharose-treated serum was prepared by passing 3 volumes ofserum through 1 volume of Lysine-Sepharose (Amersham Pharmacia Biotech)equilibrated with PBS/0.68 mM CaCl₂. Complement-grade human serum fromSigma (Cat.# S-1764) was reconstituted from lyophilized powder followingsupplier's protocol, filtered and adjusted to pH 7.4. Both rat and humansera were stored for several days on ice. CVF treatment of serum wasconducted by pre-incubating serum with CVF (10.8 μg/ml) at 37° C. for 60min.

Example 2 Complement Inactivation of Phage

[0207] Given the prominent role of complement (C) in neutralizingforeign particulate material in the blood (Sakamoto, et al., Nutrition,14:391-8. 1998), it was hypothesized that K−/R− phage were inactivatedby C deposition. Pre-injecting rats with cobra venom factor (CVF)blocked in vivo inactivation of K−/R− phage, confirming this assumption(FIG. 1B). CVF closely mimics C3 and forms an unregulated C3/C5convertase, which leads to depletion of C3 and C5 in the blood andblocks further C action (Cochrane, et al., J. Immunol., 105:55-69.1970). CVF treatment also increased recovery of the phage library fromrat serum in vtro, from less than 1% to approximately 50%. A similarincrease in phage recovery (40-60%) was obtained in vitro withindividual K−/R− clones. Thus, the phage was inactivated both in vivoand in vitro through C activation. Furthermore, the phage recovery invitro was increased to approximately 70% by addition to serum of 10 mMMg/EGTA; suggesting C activation through the classical pathway(Forsgren, et al., J. Lab. Clin. Med., 85:904-12. 1975).

[0208] The phage library was also inactivated by human serum. Theincubation of 10⁷ pfu of the phage library with 100 μl of human serum at37° C. for 30 min typically resulted in phage recovery of less than 1%.The phage recovery increased to approximately 40% and 80% aftertreatment with CVF and Mg/EGTA, respectively. CVF-sensitive inactivationof the phage library was also observed in mice (manuscript inpreparation). Therefore, the inactivation of T7 display phage by C is acommon phenomenon for different species. Human serum was different fromrat serum in that it inactivated K⁺/R+ phage as efficiently as K−/R−phage (phage recovery<1%).

Example 3 Inactivation of Phage Requires Natural Antibodies

[0209] C activation through the classical pathway is typically triggeredby formation of multivalent antigen-antibody complexes (Forsgren, etal., J. Lab. Clin. Med., 85:904-12. 1975). The role of antibodies in T7phage inactivation was determined by depleting serum of specific classesof immunoglobulins. The survival rate of phage library (10⁷ pfu) in theserum depleted of IgM under routinely used conditions was 62±16%(mean±standard deviation, n=3), as compared to 1.1±0.3% in non-treatedserum. The survival of phage in IgG-depleted serum increased to 16±4%and the depletion of IgA had no effect on the phage survival rate(0.9±0.3%). The phage survival rate increased to 80-90% when morevigorous immunodepletion of IgM was conducted.

[0210] The addition of depleted IgM back to the IgM-depleted serumsignificantly restored the ability of the serum to inactivate phagelibrary. Only 14±5% of the phage survived under these conditions. Theinactivation of phage was restored even more efficiently, decreasing thephage survival rate to 4±3%, with human plasma IgM from Calbiochem(Cat.# 401799). The IgM was isolated, according to the manufacturer, bygel-filtration followed by DEAE chromatography at pH 6.8. IgM was elutedfrom DEAE-Sepharose by gradually decreasing the pH to 5.0 (Jehanli andHough, J. Immunol. Methods, 44:199-204. 1981). Similarly prepared humanmyeloma IgM (Calbiochem Cat.# 401108) had no effect as the phagesurvival rate in reconstituted serum remained above 60%. Human plasmaIgM from Sigma (Cat.# I-8260) or Chemicon (Cat.# AG722) did not restorephage inactivation either. The corresponding phage survival rates were68±13 and 74±10%, respectively.

[0211] The 2-D electrophoresis (O'Farrell, J. Biol. Chem., 250:4007-21.1975) revealed that Calbiochem IgM had a wider range of isoforms and wasfar more acidic than Chemicon IgM. Non-reducing SDSpolyacrylamide/agarose electrophoresis (Fazel, et al., Int. Immunol.,9:1149-58. 1997) showed that both preparations contained predominantlypentameric IgM. Fractionation of Calbiochem IgM on Sephacryl 300 HR orSephacryl 400 HR (Amersham Pharmacia Biotec) demonstrated that thereconstitution activity was associated with the major IgM peak. Similarresults were obtained with the IgM fractionated by ammonium sulfateprecipitation or ion exchange chromatography on DEAE-Sepharose (data notshown). Therefore, the protein that restored phage inactivation activityto IgM-depleted serum appeared to be an acidic, predominantly pentamericform of IgM (Niles, et al., Proc. Natl. Acad. Sci., USA, 92:2884-8.1995). The role of IgM in T7 inactivation was confirmed by comparingphage survival in vivo in C57BL/6J (control) and C57BL/6J Rag-1(IgM-deficient) mice pre-injected with GdCl₃. The survival of phage in30 min after injection (10⁸-10⁹) was 20-50 times higher in C57BL/6JRag-1 mice than in C57BL/6J mice.

[0212] The effect of IgM or IgG depletion on phage inactivationindicated that the phage inactivation was mediated by natural antibodieswhose appearance in the blood does not require the presence of thecorresponding antigen (Lacroix-Desmazes, et al., J. Immunol. Methods,216:117-37. 1998). Natural antibodies are represented by IgM(Lacroix-Desmazes, et al., J. Immunol. Methods, 216:117-37. 1998) and,to a smaller extent, by IgG (Yu, et al., J. Immunol., 157:5163-8. 1996).At least two IgM (or IgG) subunits must be simultaneously engaged in theantigen binding to initiate C activation (Cooper, Adv. Immunol.,37:151-216. 1985).

[0213] Methods for Serum immunodepletion and reconstitution: Human serumwas depleted of IgM, IgG or IgA by passing 1 ml of serum through 2 ml ofSepharose containing immobilized goat anti-human IgM (μ-chain-specific;Sigma Cat.# A-9935), Protein G (Sigma Cat.# P-4691) and goat anti-humanIgA (α-chain-specific; Sigma Cat.# A-2691), respectively. The serum waseluted from the columns with PBS and collected in the total volume of 2ml. IgM was eluted from the column by Pierce Gentle Elution Buffer,dialyzed against PBS and concentrated using Centricon-100 units. Thesurvival of phage in immunodepleted serum was evaluated after incubating10 μl of phage library (10⁷ pfu) with 200 μl of serum at 37° C. for 30min. The survival of phage in reconstituted serum was assessed afterincubating 10 μl of phage library (10⁷ pfu) with 200 μl of IgM-depletedserum in the presence of exogenous IgM (0.5 mg/ml). The total samplevolume was adjusted to 400 μl with PBS/0.8 mM CaCl₂.

Example 4 IgM Initiates Phage Inactivation by Binding to DisplayedPeptides

[0214] To find the protein 10B determinant that bound IgM and caused Cactivation, we analyzed the survival in human serum of a series of phageclones with truncated 10B proteins (described in the legend to Table 3).Clone 20-6 had the shortest protein 10B and a high efficiency ofsurvival in human serum (Table 3A). Using the approach described belowfor identification of the plasma protein that protects K+/R+ phageagainst C (FIG. 2C) we found that immobilized phage 20-6 did not bind asignificant amount of any protein (data not shown). Nor was theresistance of clone 20-6 to C inactivation due to a particular primarystructure of its 10B protein carboxy-terminus. We have found no cloneswith detectable resistance to C that would display peptides similar instructure to the clone 20-6 10B protein carboxy-terminus. The phage 20-6coat protein 10B was, therefore, intrinsically resistant to C. TABLE 3Interaction of protein 10B truncated carboxy-termini with IgM in humanserum. Coat protein % Phage % Phage Resistant to Clone carboxy-terminusSurvival* Immunoprecipitation* A. 20-6 AAGAVVFQ 90 ± 9 90 ± 25 B. Wild-AAGAVVFKVE 34 ± 6 126 ± 11  C. 32-77 AAGAVVFQS 103 ± 8  86 ± 16 D. 32-23AAGAVVFSQV 54 ± 2 89 ± 9  E. 32-56 AAGAVVFQSE <0.1 22 ± 3  F. 15-28AAGAVVFQSGAA <0.1  3 ± 3  G. Display AAGAVVFQSGVM <1    4 ± 2 LGDPNSDGA(X)₁₁₄ # Immunoprecipitation samples contained 10 μl of phage(˜10⁵ pfu), 2 μl of human serum, 10 μl of 20 mM EDTA and 18 μl of PBS.EDTA was used to block C activation (Forsgren, A., et. al. J. Lab. Clin.Med. 85, 904-912 (1975)). The samples were incubated at 37° C. for 30min and then treated with 200 μl of immobilized (50% agarose slurry)goat anti-human IgM (Sigma Cat.# A-9935) or anti-human IgA (Sigma Cat.#A-2691) antibodies at 4° C. # for 30 min. Agarose was precipitated bylow-speed centrifugation and the percentage of phage resistant toimmunoprecipitation was determined by titering phage in thesupernatants. The recovery values for the phage treated with anti-IgMantibodies were normalized to the corresponding values obtained withanti-IgA antibodies used as control. The absolute recovery of phagetreated with anti-IgA antibodies was around 100%.

[0215] About 90% of the coat protein in wild-type phage is representedby protein 10A and only 10% by protein 10B. The wild-type protein 10Acan be viewed as a protein 10B in clone 20-6 that has thecarboxy-terminal Q substituted for KVE (Table 3B). Wild type T7 phagewas quite resistant to C inactivation in human serum (Table 3B).

[0216] Clone 32-77 had one additional amino acid residue at the 10Bprotein carboxy-terminus relative to clone 20-6 and showed a similarefficiency of survival in human serum (Table 3C). Clone 32-23 (Table 3D)was different from clone 20-6 in that it had one additional amino acidresidue (S) inserted between F and Q residues and another (V) located atthe carboxy-terminus. Clone 32-23 survived in serum quite well, althoughless efficiently than clones 20-6 and 32-77 (Table 3D). Clone 32-56(Table 3E) had two additional amino acid residues at thecarboxy-terminus of 10B protein relative to clone 20-26 and was almostcompletely inactivated in human serum (Table 3E). The stronginactivation of clone 32-56 was somewhat unexpected, as it was isolatedafter 4 rounds of selection for survival in human serum. The 10B proteinin clone 15-28 (Table 3F) had two additional amino acid residues (SG)from the protein 10B sequence followed by three others (AAR) of unknownorigin. This clone was inactivated in human serum (Table 3F).

[0217] Thus, the appearance of phage sensitivity to C correlated withminor changes in the structure of the protein 10B carboxy-terminus. Inparticular, the addition of as few as two amino acid residues (clone32-56) to the protein 10B carboxy-terminus in C-resistant clone 20-6 wassufficient to render this phage C-sensitive. This is consistent with thefact that all tested K−/R− clones with non-truncated coat proteins wereliable to C inactivation in rat serum. The survival of clones withtruncated 10B proteins in serum may be explained by insufficientexposure of the C-termini of these proteins to the plasma constituentsrequired for phage inactivation.

[0218] The minor changes in the structure of the protein 10Bcarboxy-terminus that rendered the phage liable to C inactivation alsocaused the recognition of phage by IgM. The efficiency ofimmunoprecipitation for C-resistant clones 20-6, 32-77 and 32-23 andwild-type phage in the samples treated with immobilized anti-IgM wasalmost as low as that in control samples treated with anti-IgA (Table 3,A-D). In contrast, the efficiency of immunoprecipitation with anti-IgMfor C-sensitive clones 32-56 and 15-28, as well as for the phage libraryin general, was quite significant (Table 3, E-G). Therefore, theinactivation of phage by C was apparently mediated by binding of IgM tothe carboxy-terminal sequence of the protein 10B.

Example 5 Peptide-Specific Natural Antibodies

[0219] The basis for IgM binding to disparate 10B proteincarboxy-terminal sequences in different clones was elucidated bystudying the ability of phage inactivated by UV-irradiation to rescuethe phage with identical or different displayed peptides from Cinactivation. For example, the recovery of phage IL-14 (Table 1D andFIG. 1A, K−/R− phage) in vivo dramatically increased if the phage (10⁹pfu) was co-injected with an excess (10¹² pfu) of the same phageinactivated with UV irradiation (FIG. 1B, IL-14/UV IL-14). TheUV-inactivated phage IL-14 did not, however, rescue phage IL-16 (Table1D and FIG. 1B, IL-16/UV IL-14). Other tested combinations of clones(Table 1D) gave results very similar to those shown in FIG. 1B. Inaddition to IL-16, UV-inactivated IL-14 did not rescue IL-1 and IL-20.UV-inactivated IL-1 rescued IL-1 but did not rescue IL-14 and IL-16.Similarly, UV-inactivated IL-21 rescued IL-21 but did not rescue IL-8and IL-32. UV-inactivated phage also rescued live phage with identicalbut not with different peptides in human serum (data not shown).

[0220] Moreover, most phage with the peptides that differ from eachother only in one amino acid residue will compete with each other in theinactivation assay only to a very limited extent (Table 4). TABLE 4Variable Amino Survival of Live Acid Residue UV Phage Live Phage Phage,% position Peptide Peptide (mean ± SD) −1 DGAI DGAI 89 ± 24 −1 DGAI DGAA 4 ± 2  −2 DGALAS DGALAS 103 ± 5  −2 DGALAS DGALSS 25 ± 22 −2 DGADLDGADL 82 ± 1  −2 DGADL DGANL  5 ± 6  −3 DGAGVY DGAGVY 74 ± 15 −3 DGAGVYDGALVY 20 ± 12

Example 6 A Serum Factor Prevents K+/R+ Phage Inactivation

[0221] Serum passed through Lysine-Sepharose (that mimics peptidecarboxy-terminal K residues (Deutsch and Mertz, Science, 170:1095-6.1970) efficiently inactivated K+/R+ phage (Table 2, B and E), suggestingthat K+/R+ phage was protected against C by a serum compound bound tocarboxy-terminal K or R amino acid residue. The protective compound waseluted from Lysine-Sepharose by either 0.5 M NaCl or 2 mM EDTA, asjudged from the restoration of the serum protective activity withrespect to K+/R+ phage by these eluates (Table 2, C and F). As expected,Lysine-Sepharose also bound plasminogen (Deutsch and Mertz, Science,170:1095-6. 1970). Plasminogen was eluted by ε-aminocaproic acid (ε-ACA)(Deutsch and Mertz, Science, 170:1095-6. 1970) and had no protectiveeffect on phage (data not shown).

Example 7 Identification of the Serum Protective Factor

[0222] The only major polypeptide eluted by 0.5 M NaCl or 2 mM EDTA hada molecular weight of approximately 30 kDa (FIG. 2A, lanes 1 and 2,respectively). The polypeptide was identified by microsequencing(Kendrick Laboratories, Madison Wis.) as CRP that is normally present inrat serum at the concentration of 0.3-0.5 mg/ml (de Beer, et al.,Immunology, 45:55-70. 1982). Immunoblot analysis confirmed that theeluted protein was CRP (FIG. 2B, lane 1).

[0223] The binding of CRP to Lysine-Sepharose could be explained by acertain similarity between the lysine residues attached to Sepharosethrough lysine α-amino groups and the predominant CRP ligand,phosphorylcholine (PC). Like PC, the carboxy-terminal lysine containstwo oppositely charged compact groups separated by a short aliphaticchain. The carboxy-terminal arginine shares this likeness. CRP is elutedfrom Lysine-Sepharose as a sharp peak with 1 mM PC in PBS/0.68 mM CaCl₂,which lends support to this notion (data not shown). The elution of CRPby EDTA from Lysine-Sepharose is consistent with the strictCa²⁺-dependence of CRP-PC interaction (Volanakis and Kaplan, Proc. Soc.Exp. Biol. Med., 136:612-4. 1971).

[0224] PC reduced the protection of K+/R+ phage both in vivo and invitro, thereby confirming the role of CRP as a K+/R+ protection agent(FIG. 1B and Table 2K, respectively). The IC₅₀ determined for K+19/16and R+19/14 clones in vitro was approximately 50 μM. Direct interactionbetween CRP and K+/R+ phage was shown with the phage immobilized onAffi-Gel 15. Immobilized K+/R+ phage, but not K−/R− phage, bound asubstantial amount of serum protein that was eluted from the column byε-ACA (FIG. 2C). SDS-PAGE showed that the major protein bound to K+ orR+ phage had the electrophoretic mobility identical to that of CRP (FIG.2A, lanes 3 and 4). The identification of this protein as CRP wasconfirmed by the Western blot analysis (FIG. 2B, lanes 2 and 3). Nobinding of CRP to K−/R− phage or a mock column was detected. The bindingof CRP to K+/R+ phage was Ca²⁺-dependent and did not take place in thepresence of Mg²⁺ alone (data not shown). The presence of multiple CRPmolecules on surface of K⁺/R+ phage could provide steric protectionagainst C-mediated inactivation. The role of CRP in protecting K+/R+phage against C is consistent with the lack of protection for K+/R+phage in human serum which normally contains little CRP.

[0225] The concentration of CRP in many species, humans included, isdramatically increased as a result of an acute phase reaction (Szalai,et al., Immunol. Res., 16:127-36. 1997). The exact role of CRP in vivois unclear. The functions ascribed to CRP include modulation of theimmune cell behavior, participation in killing infectious agents andclearance of cellular debris (Szalai, et al., Immunol. Res., 16:127-36.1997). The ligands that competitively interact with the CRP PC-bindingsites include phosphate monoesters (Volanakis and Kaplan, Proc. Soc.Exp. Biol. Med., 136:612-4. 1971), certain galactans (Volanakis andNarkates, J. Immunol., 126:1820-5. 1981, Culley, et al., J. Immunol.,156:4691-6. 1996), lipoproteins and lipids (Pepys, et al., Int. Rev.Exp. Pathol., 27:83-111. 1985), immobilized laminin and fibronectin(Tseng and Mortensen, Exp. Cell. Res., 180:303-13. 1989) and cationicpolymers and proteins (Dougherty, et al., Mol. Immunol., 28:1113-20.1991, Du Clos, et al., J. Biol. Chem., 266:2167-71. 1991). Specificbinding of CRP in situ to snRNPs (Pepys, et al., Clin. Exp. Immunol.,97:152-7. 1994) and covalent binding in vivo to C components C3 and C4have been reported (Wolbink, et al., J. Immunol., 157:473-9. 1996).

[0226] Carboxy-terminal K and R residues are readily generated in vivoat the cell or extracellular matrix surface by trypsin-like proteases(Liotta, et al., Cancer Res., 41:4629-36. 1981). The binding of CRP tosuch carboxy-termini could modulate the blood exposure of new proteinepitopes created by extensive proteolysis. The CRP binding toproteolytic products might also play a role in the clearance of cellulardebris released from dying cells (Du Clos, et al., J. Biol. Chem.,266:2167-71. 1991, Pepys, et al., Clin. Exp. Immunol., 97:152-7. 1994,Du Clos, et al., J. Immunol., 141:4266-70. 1988, Jewell, et al., Mol.Immunol., 30:701-8. 1993).

[0227] Discussion Concerning CRP's Role in Phage Protection: CRP belongsto the pentraxin family of pentameric proteins highly conserved amongdifferent mammalian species. It was originally named because it bindsthe C-polysaccharide of pneumococcus and it was eventually discoveredthat it binds the phosphocholine moiety on the C-polysaccharide. Itrequires calcium for this binding. CRP can bind the phosphocholineheadgroups of phospholipids such as phosphatidylcholine or sphingomyelinbut not in “intact” bilayers (Richards, et al., Proc. Natl. Acad. SciUSA, 74:5672-5676. 1977). Galactosyl residues in the presence ofaccessible phosphocholine groups enhance CRP binding, C1q binding, andC4 activation, which raises concerns about using galactosyl groups forhepatocyte targeting of liposomes (Volanakis and Narkates, J.Immunology, 126:1820-1825. 1981). Similarly, exposure of galactosylgroups on erythrocyte membranes by desialation activates C by CRPbinding (Pepys and Baltz, Advances in Immunology, 34:141-212. 1983).

[0228] Human CRP is a major acute phase reactant that is widely used fordetecting inflammation- and injury-related conditions (Szalai, et al.,Immunologic Research, 16:127-36. 1997). CRP is constitutively elevatedin rats. The exact role of CRP in vivo is unclear. The functionsascribed to CRP include modulation of immune cell behavior,participation in killing infectious agents and clearance of cellulardebris (Szalai, et al., Immunologic Research, 16:127-36. 1997) (Bama, etal., Cancer Research, 44:305-310. 1984). The ligands that competitivelyinteract with the CRP PC-binding site include phosphate monoesters(Volanakis and Kaplan, Proceedings of the Society for ExperimentalBiology & Medicine, 136:612-4. 1971), certain galactans (Volanakis andNarkates, J. Immunology, 126:1820-1825. 1981, Culley, et al., Journal ofImmunology, 156:4691-6. 1996), lipoproteins and lipids (Pepys, et al.,International Review of Experimental Pathology, 27:83-111. 1985),immobilized laminin and fibronectin (Tseng and Mortensen, ExperimentalCell Research, 180:303-13. 1989) and cationic polymers (polylysine,protamine, poly-arginine) and proteins (Du Clos, et al., Journal ofBiological Chemistry, 266:2167-71. 1991, Dougherty, et al., MolecularImmunology, 28:1113-20. 1991) (Siegel, et al., J. Exp. Med.,142:709-721. 1975). Thus, CRP could have important effects on a numberof non-viral vector systems. Specific binding of CRP in situ to snRNPs(Pepys, et al., Clinical & Experimental Immunology, 97:152-7. 1994) andcovalent binding in vivo to C components C3 and C4 have been reported(Wolbink, et al., Journal of Immunology, 157:473-9. 1996).

[0229] The exact mechanism by which CRP protects Lys+/Arg+ phage againstC-mediated inactivation remains to be established. Simple stericprotection could be achieved just due to the presence of multiple CRPmolecules on the phage surface. Bound CRP may also directly inhibit Caction at the phage surface. Although CRP activates the C classicalpathway (Kaplan and Volanakis, Journal of Immunology, 112:2135-47. 1974,Siegel, et al., Journal of Experimental Medicine, 140:631-47. 1974), italso inhibits the activation of the alternative and lectin pathways onthe surface to which it binds. The inhibition is associated withincreased C regulatory protein H binding to C3b (Suankratay, et al.,Clinical & Experimental Immunology, 113:353-359. 1998, Mold, et al., J.Immunol., 133:822-825. 1984). Perhaps, CRP's inactivation of thealternative pathway is predominant and protects the phage from Cinactivation.

[0230] An important aspect of the finding that protein C-reactiveprotein binds to C-terminal Lys and Arg residues is a frequentoccurrence of such C-termini in the blood. They are generated at thecell or extracellular matrix surface by blood trypsin-like proteasessuch as thrombin and plasmin (Liotta, et al., Cancer Research,41:4629-36. 1981). The binding of CRP to such C-termini could regulatethe interaction of C and immune system cells with potentiallyimmunogenic new epitopes (Szalai, et al., Immunologic Research,16:127-36. 1997, Suankratay, et al., Clinical & Experimental Immunology,113:353-359. 1998). The CRP binding to proteolytic products might alsoplay a role in the clearance of nuclear debris released from dying cells(Du Clos, et al., Journal of Biological Chemistry, 266:2167-71. 1991,Pepys, et al., Clinical & Experimental Immunology, 97:152-7. 1994, DuClos, et al., Journal of Immunology, 141:4266-70. 1988, Jewell, et al.,Molecular Immunology, 30:701-8. 1993). The abundance of Lys and Arg innuclear proteins makes proteolytic generation of C-terminal Lys and Argaa particularly easy. It is noteworthy that the nuclear protein Sm-Dthat binds to CRP in solution (Jewell, et al., Molecular Immunology,30:701-8. 1993) and is a constituent of snRNPs recognized by CRP in situ(Pepys, et al., Clinical & Experimental Immunology, 97:152-7. 1994) hasa Lys at the C-terminus. A C-terminal Lys is also present in histone H1that is required for CRP binding to chromatin in vitro (Du Clos, et al.,Journal of Immunology, 141:4266-70. 1988). It should also be mentionedthat all of the peptides that corresponded to nuclear protein sequencesand bound to CRP in vitro incidentally contained a Lys or Arg at theC-terminus (Du Clos, et al., Journal of Biological Chemistry,266:2167-71. 1991, Jewell, et al., Molecular Immunology, 30:701-8.1993).

[0231] The observed selection of exclusively Lys+/Arg+ clones resultedfrom the combination of used selection conditions and make-up of theoriginal library. Lys+/Arg+ clones were initially present in the librarysince there was no pre-selection against truncated peptides resultingfrom stop-codons. The selection of Lys+/Arg+ clones was promoted as wellby macrophage suppression and a long phage circulation time. Usingdifferent conditions, we were able to select Lys−/Arg− phage thatpersisted in vivo (see below). Thus, the T7 display system used hereinappears to have wide applicability with respect to selecting peptidedeterminants recognized by blood proteins in vivo.

[0232] Methods for Isolation and testing of CR1P. 100 ml of filtered,Sprague-Dawley rat serum (Pel-Freez Biologicals) was applied onto a25-ml column of Lysine-Sepharose equilibrated in PBS/0.68 mM CaCl₂. Thecolumn was washed with 10 volumes of PBS/0.68 mM CaCl₂ and CRP waseluted with 2 mM EDTA in PBS. CRP was dialyzed against PBS, concentratedand stored at −20° C. CRP-enriched protein fraction eluted by 0.5 M NaClwas prepared using a plasminogen isolation protocol 21.

[0233] Methods for Affinity isolation of the serum protein protectingK+/R+ phage against Phage was immobilized on Affi-Gel 15 (Bio-Rad)following supplier's protocol. 10¹³ pfu of phage in 3 ml of 50 mM MOPS(pH 7.5) were incubated with 4 ml of settled gel overnight at 4° C.under constant mixing. The column was washed with 10 volumes of 10 mMTris-HCl/1M NaCl/1 mM EDTA/ (pH 8.0) and 10 volume of PBS. 1 ml ofsettled gel contained 60-120 μg of phage protein. Rat serum (3 ml) wasapplied onto a column (4 ml) equilibrated in PBS/0.68 mM CaCl₂. Thecolumn was washed with PBS/0.68 mM CaCl₂ (8 volumes) and phage-boundprotein was eluted with 2-20 mM ε-ACA in PBS/0.68 CaCl₂. 1.5 mlfractions were collected. Protein was determined by a BCA assay(Pierce). The amount of eluted protein was normalized to the amount ofphage protein on the column, taking R+ phage as a standard.

Example 8 Selection for Phage Persisting in Human Serum

[0234] To compare the survival strategies of phage in sera fromdifferent species, we also selected for phage that survived in humanserum. 10⁹ pfu of phage library (50 μl) was incubated with 1 ml of humanserum (pH 7.4) at 37° C. for 30 min and amplified as described above.

[0235] The phage survival rate in the 1^(st) round of selection was lessthan 1%. The survival rate rose to about 10% in the 2^(nd) round andreached approximately 35% in the 5^(th) round of selection. Thesequencing after the 1^(st) round of selection revealed a much higherthan expected (24 vs.3%, respectively) portion of clones with tyrosine(Y) residues in displayed peptides (Y+ clones; Table 1E). Other peptidesselected in the 1^(st) round of selection showed no similarity and werenot included in further analysis.

[0236] All Y+ clones showed a relatively high rate of survival in humanserum. The survival rate for different Y+ clones (Table 1F) was in therange of 25 to 60%. Sequencing of the clones after the 3^(d) and 4^(th)round of selection showed only a marginal increase in the percentage ofY+ clones (˜30%), presumably due to a competing increase in the portionof clones with truncated 10B proteins (Table 3).

[0237] The differences in the structure of selected Y+ peptidessuggested that the phage was protected against C via binding to plasmaproteins rather than through assuming a specific “cryptic” conformationthat is not recognized by natural antibodies. We found in ourpreliminary experiments with immobilized Y+ phage (clone 32-5, Table 1E)that the phage bound α₂-macroglobulin in human serum. Consistent withthis observation, the survival rate for selected Y+ clones in rat serum,that has a much lower level of α₂-macroglobulin, was approximately5-10-fold lower than in human serum.

Example 9 Selecting Phage T7 Display Clones Persisting in the Blood ofRats not Treated with Gadolinium

[0238] The phage resistant to inactivation by C was selected survivingin rats with intact macrophages for 60 min. 15 individual selectedclones were sequenced and all of them found to have the 10B proteintruncated after Q₃₄₃ ⁹ as a result of a single nonsense mutation in theserine codon (TCA

TAA). The selected phage did not display peptides as the mutationoccurred upstream of the peptide cloning site. The sequenced clonesbelonged to three different genotypes, as judged from the different DNAsequences at the peptide cloning site. All clones were remarkably stablein the blood. Both with and without Gd pre-injection, the efficiency ofthe live phage recovery from plasma remained at the same level, in therange of 80-100%, for at least 30 min after phage injection.Approximately 4% of the injected phage could be recovered from the liverwhen the animals were perfused 5 min after phage injection. The presenceof phage in the liver might be due to incomplete perfusion. The spleen,lungs and kidneys showed only trace phage accumulation. The persistenceof clones with different genotypes in the blood at the same levelpointed to the importance of the 10B protein truncation shared by allclones for phage survival. The contribution of other, undetectedmutations into phage survival could not be ruled out but, even ifpresent, was very limited. Neither in these nor in all followingexperiments did we detect any phage with a significant rate of survivaland no obvious changes in the carboxy-terminal portion of the 10Bprotein. Therefore, the inactivation of the T7 phage library in rats wasalmost exclusively, if not entirely, driven by the structure of the 110Bprotein carboxy-terminus.

[0239] Affinity purification of an IgM species that bind specific T7phage. Experimental design: To elucidate more exactly the role of IgM inpeptide-dependent phage inactivation, we will study if there are indeeddifferent classes of IgM that recognize different peptides. Analternative might be the existence of some other proteins that binddifferent classes of peptides and interact with the same class of IgMthat activates complement. The specificity of an IgM species will alsobe confirmed.

[0240] Methods: T7 phage from the clone IL-14 (DGAKIPY) and two otherclones, IL-13 (DGAVAYPPMLPVLHGSLARL) and IL-20 (DGAYNAKTDRG), that werenot protected by an excess of UV-inactivated IL-14 will be immobilizedon pre-activated Affi-Gel-10 by incubating phage with Affi-Gel overnightat 4° C. with end-over-end mixing. The resin will be washed withhigh-salt phage extraction buffer followed by Pierce Gentle ElutionBuffer to eliminate a free phage contamination. Human or rat serumadjusted to the pH of 7.4 will be passed through the column withimmobilized phage at 4° C. and the column will be washed with 5-10volume of cold PBS containing. The serum proteins that bound to theimmobilized phage will be eluted with Pierce Gentle Elution buffer. Theeluate will be concentrated by centrifugation in Centricon 30 units anddialyzed against PBS. The resultant solution will be applied onto theaffinity column containing goat antibodies (IgG) against human IgM. Thecolumn will be washed with 10 volume of PBS/0.5 M NaCl and IgM will beeluted with Pierce Gentle elution buffer. The eluted IgM will beconcentrated, dialyzed against PBS and added to IgM-depleted serum totest its ability to reconstitute the inactivation by the serum of thephage clone that was used as an affinity ligand in the IgM isolation ascompared to the phage clones with different peptides. Thus, thespecificity of a particular IgM species will be determined.

[0241] Biotin Inactivation Methods

[0242] Biotinylation of T7 Phage: We have developed a novel system forselecting for phage that has been (Table 5) TABLE 5 Infectivity ofbiotinylated T7 phage and its inactivation by neutravidin. ConcentrationTotal number of phage Inhibition of of Biotin- Neutravidin plaquesnormalized to plaque-forming LC-NHS, mM treatment phage dilutionactivity, % 0 23,800 − − 0.2 − 19,600 0.2 + 72 99.63 0.5 − 15,600 0.5 +0 100

[0243] internalized in cells. The idea is to lightly biotinylate thephage and inactivate it with neutravidin. If the phage has beeninternalized then it will be inaccessible to neutravidin and remaininfectious. T7 phage can be biotinylated with only a small loss in itsinfectivity and this infectivity can be suppressed by neutravidin. T7was labeled with Biotin-LC-NHS (Pierce), incubated with neutravidin(Pierce) and plated as recommended by the manufacturer (Novagen). Wefound that the interaction between the phage surface-conjugated biotinand free neutravidin can be used as an efficient inactivation “switch”with T7 phage display libraries. The treatment of T7 with 0.5 mMBiotin-LC-NHS resulted in very strong inhibition of phage infectivity byneutravidin. The inhibition of phage plaque-forming activity caused bythe labeling procedure itself was 30-35% (for 0.5 mM Biotin-LC-NHS).

[0244] To show the accessibility of non-internalized,endothelium-attached phage to neutravidin, we used leg muscle as amodel. After intravascular injection of biotinylated T7 phage library,the leg muscle was perfused with either PBS or PBS+ neutravidin. Inmuscles that were perfused with neutravidin, 80% of muscle bound phagewas inactivated.

[0245] Determination of whether peptides with amino termini invoke thesame process. Experimental design: The peptides displayed on T7 phageare cloned into the C-terminal portion of the phage coat protein 10Band, therefore, have free C-termini. In order to study the interactionwith serum of the peptides that have free N-termini, we will use peptidelibraries displayed on phage M13. In M13 peptide libraries, the peptidesare cloned into the N-terminal portion of the phage coat proteins pIIIor pVIII.

[0246] Methods: M13 libraries that display peptides in pIII (Bio-Labs)or pVIII (courtesy of G. Smith, University of Missouri) proteins andthat have complexity around 10⁹ are used. Commercially availablelibraries from New England Bio-Labs have 7 and 12 amino acid residuelong peptides. The f88-4 library from the Smith lab has 15 amino acidresidue long peptides in pVIII (appr. 300 copies per phage particle).The libraries are incubated in mouse serum essentially as describedabove for T7 phage and the surviving phage is measured by a platingassay.

[0247] All tried M13 phage display libraries are inactivated by bothhuman and rat serum. 10⁶ pfu of phage is inactivated by 100 μl of serumduring the incubation at 37° C. for 30 min by 98-99%. A co-incubation oflive phage with UV-inactivated phage (10¹⁰ pfu prior to inactivation) ofthe same type rescues the majority of live phage.

[0248] The role of displayed peptides in phage inactivation isdetermined by co-incubating live library with UV-inactivated vectorphage or wild-type M13 phage. The phage inactivation caused by therecognition of displayed peptides by specific natural antibodies can notbe offset by the presence of an excess of wild-type phage coat proteins.The rescue of phage with displayed peptide by an excess of inactivatedwild-type phage indicates that the phage is inactivated due to theinteraction of serum constituents with wild-type proteins. On the otherhand, the rescue of phage with displayed peptide by an excess ofinactivated phage with displayed peptides only indicates that the phageis inactivated due to the interaction of serum constituents withdisplayed peptides.

[0249] Prolonged Circulation of Lys+/Arg+ Phage in Rats not Treated withGadolinium

[0250] Although the Lys+/Arg+ phage were obtained by performing theselections in rats treated with gadolinium, they had significantlyprolonged circulation in rats not treated with gadolinium. Severalpercent of injected Lys+/Arg+ phage could be recovered from rat plasmain an infectious state 5 min after injection. Almost no infectiousLys−/Arg− phage was recovered from plasma under these conditions.

[0251] Although, the actual circulation time of lys+/arg+ phage isrelatively short compared to sterically-stabilized liposomes, theirprolonged circulation is very significant for the following reasons.One, the lys+/arg+ phage have a blood circulation time that is more than1,000-fold above the non-selected phage given that almost all of thephage is inactivated by 5 minutes. In comparison, PEG increases thecirculation times of liposomes approximately hundred-fold. One needs toappreciate that the baseline for phage inactivation is different thanthat for liposomes which are inherently more stable in the blood thanphage. Two, the fast, almost instant inactivation of most T7 clones inblood is, in fact, a very important feature of our selection system asit provides low background and short incubation times for selection ofweak protein-phage interactions. Three, a relatively short circulationtime of CRP-protected phage appears to be due to non-specific,concentration-independent phage inactivation/clearance. A thousand-foldincrease in the amount of injected Lys+ phage had no effect on thepercentage of live phage in the blood. In contrast, a thousand-foldincrease in the amount of injected Lys− (not recognized by CRP) phageresulted in a dramatic increase in the survival of this phage in theblood. The survival of Lys− phage under these conditions wasquantitatively very similar to that of Lys+ phage. Further increase inthe amount of Lys− injected phage had no effect on the survivalpercentage. These results indicate that the circulation time of ourselected phage is quite close to the maximum that can be obtained withthis system. Artificial delivery vectors bearing the lys+/arg+ peptidesmay not be subject to this non-specific inactivation/clearance.

[0252] Selection of Phage Clones Resistant to Human ComplementInactivation

[0253] The binding and precipitating properties of CRPs from differentspecies show certain. Further studies are necessary to extend ourinitial findings in the rat serum to human serum. “Lysine−/arginine−”phage are 100% inactivated by “normal” human serum containing just traceamounts of CRP. The extent of inactivation by human serum is comparableto that by rat serum Therefore, the inactivation of T7 phage by serum isa universal phenomenon, not confined to a particular species. Sincehuman serum is naturally low in CRP (<10 μg/ml except during an acutephase response), we reasoned that selections in human serum would yieldserum resistant “lys−/arg−” clones containing novel peptide sequencesnot obtained from the rat studies.

[0254] Selection of peptides that confer resistance on T7 phage againsthuman complement was carried out using pooled commercial “complementgrade” serum from Sigma (Cat. # S1764) and a new T7 peptide librarycontaining 1-13 amino acid long linear peptides. The selection wasperformed under relatively “mild” selection pressure, using a relativelylarge phage/serum ratio. Lyophilized serum was reconstituted with waterand filtered through a 0.22 μm filter prior to use. 10⁹ pfu of the T7library in 200 μl of PBS were incubated with 1 ml of serum for 30 minand complement-resistant phage was amplified in a 0.5 L log culture ofBL 21 E. coli. Amplified phage was isolated and the procedure wasrepeated 2 more times using the same conditions. 30 individual cloneswere isolated from the selected phage population, sequenced and groupedaccording to certain consensus residues (Table 5).

[0255] An obvious trait shared by many selected peptides was the highfrequency occurrence of Ser and Thr residues near the C-terminus. 6clones particularly stood out in that they had double or triple cysteinsat the C-terminus. All other clones are presented in Table 5 accordingto the position of Ser/Thr residues in the corresponding peptides.

[0256] Table 5. Peptide sequences of the 30 individual clones that wereobtained after selection for resistance to inactivation by human serum.The clones were divided into A-F groups according to the followingcriteria A. Peptides with double or triple Ser residues at theC-terminus; B. Peptides with single Ser residues at the C-terminus; C.Peptides with a Ser residue at the −2 position; D. Peptides with a Serresidue at the −3 position; E. Peptides containing Ser or Thr residuesat any other but C-terminal, −2 or −3 positions; F. Peptides that do notcontain Ser and Thr residues. Ser and Thr residues are shown in bold.The linker portion of 10B protein upstream of random peptides (10B) isunderlined TABLE 5 Clones selected for resistance against inactivationby human serum A. Multiple COOH Ser B. Single COOH Ser C. Ser at −2Position 24-1 DGALSS* 24-15 DGALAS 24-3 DGANSP 24-10 DGAHSSS 24-22 DGAWS24-8 DGASSV 24-11 DGASNLSS 24-12 DGASDRGNEEMSF 24-16 DGAARNTLSS 24-21DGAAISSDGFINQSS 24-24 DGALSS* D. Ser at −3 Position E. Ser or Thr/NotCOOH F. No Ser or Thr 24-6 DGAMSPL 24-4 DGAPSLSVGG 24-2 DGAVPL 24-14DGAVPSVSSPSIG 24-5 DGATTVDNM 24-13 DGARA 24-23 DGASGPSVG 24-7DGANLVSGTRLD 24-20 DGAMVG 24-27 DGATTSLG 24-9 DGATG 24-26 DGAVRRG 24-28DGSQM 24-17 DGATTQTAY 24-29 DGAALVL 24-18 DGASNLPL 24-19 DGAATRGR 24-25DGASKKTVLAMNPR 24-30 DGATHGSEVA

[0257] To evaluate the efficiency of selection, all selected clones wereamplified and tested individually for survival in human serum.Individual clones were tested for survival in human serum by incubating10 μl of T7 phage with 300 μl of reconstituted serum diluted with 190 μlof PBS. The incubation was carried out at 37° C. for 30 min and theefficiency of phage survival was estimated by plating as describedearlier. Percentage of T7 phage recovered (X axis) equals (# ofrecovered phage/# of injected phage) X 100. 10⁷ PFU's were used. Wefound that all selected clones with double Ser residues at theC-terminus showed a significant resistance to complement inactivation(˜15 to ˜55 percent survival). Furthermore, the presence of double Serright at the C-terminus appears to be critical for phage survival. Thus,clone 24-8 that displayed peptide SSV showed a low resistance tocomplement inactivation (<1% survival) despite the presence of Serresidues at −2 and −3 positions. In addition, it is not clear whetherthe presence of a single Ser residue at the C-terminus is sufficient toprotect phage against complement. Out of two selected clones with asingle Ser residue at the C-terminus, one was quite resistant to seruminactivation (24/15) while the other (24/22) was totally inactivated. Itis necessary to examine further clones to determine the role of otherresidues on the ability of a single C-terminal Ser to confer resistanceto human serum. A number of selected clones that showed resistance tocomplement inactivation did not have peptides with Ser residues at theC-terminus (Table 6). Most interestingly, this suggests that selectedpeptides protect phage against complement inactivation via differentmechanisms. TABLE 6 Selected peptides that confer on T7 resistance tocomplement inactivation and do not have C-terminal Ser residues.Peptides in columns A and B are grouped based on a certain amount ofsimilarity in the primary structure of C- termini. A. B. C. 24-6 DGAMSPL24-19 DGAATRGR 24-5 DGATTVDNM 24-18 DGASNLPL 24-26 DGAVRRG 24-17DGATTQTAY 24-29 DGAALVL 24-25 DGASKKTVLAMNPR 24-27 DGATTSLG 24-28 DGSQM

[0258] The exact mechanisms of phage protection by selected peptides arecurrently being studied. There are two possible mechanisms: one, thepeptide binds a serum protein that prevents C inactivation, or the twoSer residues act like PEG and reduce all serum protein interactions. Ourproposed methods of using phage affinity columns (see Exp Protocolsection) will distinguish between these two hypotheses. If it should bethe first mechanism, then the exact serum proteins that bind the phageand prevent C inactivation will be identified Oust as we did with theidentification of CRP for rat serum).

[0259] In addition, it appears that the C terminus carboxyl group playsan important role since acidification of the serum abrogates theprotection of the above selected clones. Further selections in humanserum with adjusted pH's are in progress.

[0260] Inactivation of Phage Display Library by Pup Rat Serum and theSerum From Gnotobiotic Rats.

[0261] Serum from newborn rats did not inactivate phage to a significantextent. Only 7.7+/−5.6% of the input phage was inactivated as comparedto 96.3+/−0.3% in the serum from adult rats. The inactivation of phageby the serum of new-born rats could be induced by adding to the samples100 μg of IgM (purified as described above). The efficiency of phageinactivation increased under these conditions to 70.5+/−4%. Thissuggests that the peptide-specific IgM develops in the neonatal period.Serum from gnotobiotic rats in vitro and in vivo inactivated phage aseffectively as the serum from control animals indicating that theappearance of “active IgM” is not induced by environmental agents.

[0262] Liver Targeting and Blood Persistence of T7 Phage with TruncatedProteins in Rats

[0263] We have identified a number of spontaneous mutations that occuraround the site corresponding to the translation shift point from 10A to10B protein reading frame. Most of these phage clones were selected dueto their resistance to serum inactivation. Interestingly, we discoveredthat their targeting behavior in vivo showed a wide spectrum of behavior(FIG. 1).

[0264] Both clones 20/6 (FQ*) and 32/77 (FQS*) are relatively stable inthe blood but differ substantially in their liver targeting (FIG. 1).The one additional C-terminus serine in 32/77 results in over 20% of theinjected phage ending up in the liver. Clones 32/33 (FSQV) and #112(FQSGVMLGDPN*) also target to the liver but are not as stable in blood.Clones 114 and T7 vector, shown for control purposes, are not stable inblood and can not be detected in liver liver. The overall impressionfrom these experiments is that liver targeting is associated with theexposure of peptides on the phage surface, regardless of the peptideprimary structure.

[0265] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in cell biology, chemistry, molecular biology,biochemistry or related fields are intended to be within the scope ofthe following claims.

1 105 1 10 PRT Bacteriophage T7 1 Ala Ala Gly Ala Val Val Phe Lys ValGlu 1 5 10 2 8 PRT Artificial synthetic peptide sequence 2 Ala Ala GlyAla Val Val Phe Gln 1 5 3 9 PRT Artificial synthetic peptide 3 Ala AlaGly Ala Val Val Phe Gln Ser 1 5 4 10 PRT artificial synthtic peptide 4Ala Ala Gly Ala Val Val Phe Gln Ser Glu 1 5 10 5 13 PRT Artificialsynthetic peptide 5 Ala Ala Gly Ala Val Val Phe Gln Ser Gly Ala Ala Arg1 5 10 6 35 PRT Artificial synthetic peptide 6 Ala Ala Gly Ala Val ValPhe Gln Ser Gly Val Met Leu Gly Asp Pro 1 5 10 15 Asn Ser Asp Gly AlaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa 35 7 10PRT Artificial synthetic peptide 7 Ala Ala Gly Ala Val Val Phe Ser GlnVal 1 5 10 8 13 PRT Artificial synthetic peptide 8 Ala Ala Thr Gly SerAsp Gln Gly Leu Asn Lys Ala Tyr 1 5 10 9 5 PRT Artificial syntheticpeptide 9 Ala Phe Thr Asn Lys 1 5 10 6 PRT Artificial synthetic peptide10 Ala Arg Pro Val Gln Lys 1 5 11 5 PRT Artificial synthetic peptide 11Ala Ser Arg Val Arg 1 5 12 13 PRT Simian virus 40 12 Cys Gly Tyr Gly ProLys Lys Lys Arg Lys Val Gly Gly 1 5 10 13 22 PRT Xenopus laevis 13 CysLys Lys Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 1 5 10 15Ala Lys Lys Lys Lys Leu 20 14 14 PRT Homo sapiens 14 Cys Lys Lys Lys GlyPro Ala Ala Lys Arg Val Lys Leu Asp 1 5 10 15 39 PRT Simian virus 40 15Cys Lys Lys Lys Ser Ser Ser Asp Asp Glu Ala Thr Ala Asp Ser Gln 1 5 1015 His Ser Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Asp 20 2530 Phe Pro Ser Glu Leu Leu Ser 35 16 37 PRT Simian virus 40 16 Cys LysLys Lys Trp Asp Asp Glu Ala Thr Ala Asp Ser Gln His Ser 1 5 10 15 ThrPro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Asp Phe Pro 20 25 30 SerGlu Leu Leu Ser 35 17 10 PRT Human adenovirus type 5 17 Cys Lys Arg GlyPro Lys Arg Pro Arg Pro 1 5 10 18 31 PRT Homo sapiens 18 Cys Tyr Asn AspPhe Gly Asn Tyr Asn Asn Gln Ser Ser Asn Phe Gly 1 5 10 15 Pro Met LysGln Gly Asn Phe Gly Gly Arg Ser Ser Gly Pro Tyr 20 25 30 19 4 PRTArtificial synthetic peptide 19 Asp Gly Ala Ala 1 20 15 PRT Artificialsynthetic peptide 20 Asp Gly Ala Ala Ile Ser Ser Asp Gly Phe Ile Asn GlnSer Ser 1 5 10 15 21 7 PRT Artificial synthetic peptide 21 Asp Gly AlaAla Leu Val Leu 1 5 22 10 PRT Artificial synthetic peptide 22 Asp GlyAla Ala Arg Asn Thr Leu Ser Ser 1 5 10 23 8 PRT Artificial syntheticpeptide 23 Asp Gly Ala Ala Thr Arg Gly Arg 1 5 24 5 PRT Artificialrandom library peptide 24 Asp Gly Ala Asp Leu 1 5 25 6 PRT Artificialrandom library peptide 25 Asp Gly Ala Gly Val Tyr 1 5 26 7 PRTArtificial random library peptide 26 Asp Gly Ala His Ser Ser Ser 1 5 274 PRT Artificial random libray peptide 27 Asp Gly Ala Ile 1 28 7 PRTArtificial random libray peptide 28 Asp Gly Ala Lys Ile Pro Tyr 1 5 29 6PRT Artificial random library peptide 29 Asp Gly Ala Leu Ala Ser 1 5 306 PRT Artificial random library peptide 30 Asp Gly Ala Leu Ser Ser 1 531 6 PRT Artificial random library peptide 31 Asp Gly Ala Leu Val Tyr 15 32 7 PRT Artificial random library peptide 32 Asp Gly Ala Met Ser ProLeu 1 5 33 6 PRT Artificial random libray peptide 33 Asp Gly Ala Met ValGly 1 5 34 5 PRT Artificial random library peptide 34 Asp Gly Ala AsnLeu 1 5 35 12 PRT Artificial random library peptide 35 Asp Gly Ala AsnLeu Val Ser Gly Thr Arg Leu Asp 1 5 10 36 6 PRT Artificial randomlibrary peptide 36 Asp Gly Ala Asn Ser Pro 1 5 37 10 PRT Artificialrandom libray peptide 37 Asp Gly Ala Pro Ser Leu Ser Val Gly Gly 1 5 1038 5 PRT Artificial random library peptide 38 Asp Gly Ala Arg Ala 1 5 3913 PRT Artificial random library peptide 39 Asp Gly Ala Ser Asp Arg GlyAsn Glu Glu Met Ser Phe 1 5 10 40 9 PRT Artificial random librarypeptide 40 Asp Gly Ala Ser Gly Pro Ser Val Gly 1 5 41 14 PRT Artificialrandom libray peptide 41 Asp Gly Ala Ser Lys Lys Thr Val Leu Ala Met AsnPro Arg 1 5 10 42 8 PRT Artificial random library peptide 42 Asp Gly AlaSer Asn Leu Pro Leu 1 5 43 8 PRT Artificial random library peptide 43Asp Gly Ala Ser Asn Leu Ser Ser 1 5 44 6 PRT Artificial random librarypeptide 44 Asp Gly Ala Ser Ser Val 1 5 45 5 PRT Artificial randomlibrary peptide 45 Asp Gly Ala Thr Gly 1 5 46 10 PRT Artificial randomlibrary peptide 46 Asp Gly Ala Thr His Gly Ser Glu Val Ala 1 5 10 47 9PRT Artificial random library peptide 47 Asp Gly Ala Thr Thr Gln Thr AlaTyr 1 5 48 8 PRT Artificial random library peptide 48 Asp Gly Ala ThrThr Ser Leu Gly 1 5 49 9 PRT Artificial random library peptide 49 AspGly Ala Thr Thr Val Asp Asn Met 1 5 50 20 PRT Artificial random librarypeptide 50 Asp Gly Ala Val Ala Tyr Pro Pro Met Leu Pro Val Leu His GlySer 1 5 10 15 Leu Ala Arg Leu 20 51 6 PRT Artificial random librarypeptide 51 Asp Gly Ala Val Pro Leu 1 5 52 13 PRT Artificial randomlibrary peptide 52 Asp Gly Ala Val Pro Ser Val Ser Ser Pro Ser Ile Gly 15 10 53 7 PRT Artificial random library peptide 53 Asp Gly Ala Val ArgArg Gly 1 5 54 5 PRT Artificial random library peptide 54 Asp Gly AlaTrp Ser 1 5 55 11 PRT Artificial random library peptide 55 Asp Gly AlaTyr Asn Ala Lys Thr Asp Arg Gly 1 5 10 56 5 PRT Artificial randomlibrary peptide 56 Asp Gly Ser Gln Met 1 5 57 7 PRT Artificial randomlibrary peptide 57 Asp Asn Thr Pro Lys Thr Lys 1 5 58 11 PRT Artificialrandom library peptide 58 Phe Gln Ser Gly Val Met Leu Gly Asp Pro Asn 15 10 59 27 PRT Artificial random library peptide 59 Phe Gln Ser Gly ValMet Leu Gly Asp Pro Asn Ser Asp Gly Ala Leu 1 5 10 15 Arg Gln Ser GlyArg Gly Lys Ser Ser Arg Pro 20 25 60 23 PRT Bacteriophage T7 60 Phe GlnSer Gly Val Met Leu Gly Asp Pro Asn Ser Ser Ser Val Asp 1 5 10 15 LysLeu Ala Ala Ala Leu Glu 20 61 4 PRT Bacteriophage T7 61 Phe Ser Gln Val1 62 14 PRT Artificial random library peptide 62 Gly Lys Gly Lys Thr AspAsp Pro Arg Tyr Gln Lys Phe Thr 1 5 10 63 4 PRT Artificial randomlibrary peptide 63 Gly Arg Leu Lys 1 64 6 PRT Artificial random librarypeptide 64 Gly Val Arg Glu Pro Lys 1 5 65 12 PRT Artificial randomlibrary peptide 65 His Arg Pro Lys Glu Gly Gly Lys Pro Ala Leu Lys 1 510 66 5 PRT Artificial random library peptide 66 Ile Glu Phe Ser Gly 1 567 4 PRT Homo sapiens 67 Lys Asp Glu Leu 1 68 4 PRT Artificial randomlibrary peptide 68 Lys Ile Pro Tyr 1 69 5 PRT Artificial random librarypeptide 69 Lys Leu Arg Met Lys 1 5 70 4 PRT Artificial random librarypeptide 70 Lys His Met Arg 1 71 8 PRT Artificial random library peptide71 Lys Ser Gly Gly Pro Ala Glu Arg 1 5 72 9 PRT Artificial randomlibrary peptide 72 Lys Thr Asn Val Glu Lys Gly Pro Met 1 5 73 5 PRTArtificial random library peptide 73 Lys Val Arg Glu Lys 1 5 74 5 PRTArtificial random library peptide 74 Leu Pro Ser Gly Gly 1 5 75 6 PRTArtificial random library peptide 75 Leu Ser Ala Arg Ala Pro 1 5 76 343PRT Bacteriophage T7 76 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly ThrAsn Gln Gly Lys 1 5 10 15 Gly Val Val Ala Ala Gly Asp Lys Leu Ala LeuPhe Leu Lys Val Phe 20 25 30 Gly Gly Glu Val Leu Thr Ala Phe Ala Arg ThrSer Val Thr Thr Ser 35 40 45 Arg His Met Val Arg Ser Ile Ser Ser Gly LysSer Ala Gln Phe Pro 50 55 60 Val Leu Gly Arg Thr Gln Ala Ala Tyr Leu AlaPro Gly Glu Asn Leu 65 70 75 80 Asp Asp Lys Arg Lys Asp Ile Lys His ThrGlu Lys Val Ile Thr Ile 85 90 95 Asp Gly Leu Leu Thr Ala Asp Val Leu IleTyr Asp Ile Glu Asp Ala 100 105 110 Met Asn His Tyr Asp Val Arg Ser GluTyr Thr Ser Gln Leu Gly Glu 115 120 125 Ser Leu Ala Met Ala Ala Asp GlyAla Val Leu Ala Glu Ile Ala Gly 130 135 140 Leu Cys Asn Val Glu Ser LysTyr Asn Glu Asn Ile Glu Gly Leu Gly 145 150 155 160 Thr Ala Thr Val IleGlu Thr Thr Gln Asn Lys Ala Ala Leu Thr Asp 165 170 175 Gln Val Ala LeuGly Lys Glu Ile Ile Ala Ala Leu Thr Lys Ala Arg 180 185 190 Ala Ala LeuThr Lys Asn Tyr Val Pro Ala Ala Asp Arg Val Phe Tyr 195 200 205 Cys AspPro Asp Ser Tyr Ser Ala Ile Leu Ala Ala Leu Met Pro Asn 210 215 220 AlaAla Asn Tyr Ala Ala Leu Ile Asp Pro Glu Lys Gly Ser Ile Arg 225 230 235240 Asn Val Met Gly Phe Glu Val Val Glu Val Pro His Leu Thr Ala Gly 245250 255 Gly Ala Gly Thr Ala Arg Glu Gly Thr Thr Gly Gln Lys His Val Phe260 265 270 Pro Ala Asn Lys Gly Glu Gly Asn Val Lys Val Ala Lys Asp AsnVal 275 280 285 Ile Gly Leu Phe Met His Arg Ser Ala Val Gly Thr Val LysLeu Arg 290 295 300 Asp Leu Ala Leu Glu Arg Ala Arg Arg Ala Asn Phe GlnAla Asp Gln 305 310 315 320 Ile Ile Ala Lys Tyr Ala Met Gly His Gly GlyLeu Arg Pro Glu Ala 325 330 335 Ala Gly Ala Val Val Phe Gln 340 77 5 PRTArtificial random library peptide 77 Met Ala Thr Val Lys 1 5 78 13 PRTArtificial random library peptide 78 Met Asp Ser Met Ser Asn Thr Pro AsnGly Ser Glu Arg 1 5 10 79 4 PRT Artificial random library peptide 79 MetGln Tyr Ser 1 80 10 PRT Artificial random library peptide 80 Met Val LeuPro Phe Gln Gln Thr Val Ala 1 5 10 81 5 PRT Artificial random librarypeptide 81 Met Val Arg Arg Val 1 5 82 9 PRT Artificial random librarypeptide 82 Asn Asn Ala Gln Gly Ala Arg Val Lys 1 5 83 35 PRT Artificialsynthetic peptide 83 Asn Ser Asp Gly Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ala ValLys Leu Ala Ala 20 25 30 Ala Leu Glu 35 84 9 PRT Artificial randomlibrary peptide 84 Asn Ser Asn Ala Gly Leu Glu Asn His 1 5 85 13 PRTArtificial synthetic peptide (Novagen plasmid expression sequence) 85Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu 1 5 10 86 8 PRTArtificial random library peptide 86 Pro Ser Ser Gln Gln Ala Gln Arg 1 587 4 PRT Artificial random library peptide 87 Pro Thr Ile Lys 1 88 13PRT Artificial random library peptide 88 Gln Glu Ser Arg Thr Glu Thr AspSer Gln Tyr Leu Ala 1 5 10 89 5 PRT Artificial random library peptide 89Gln Gly Asp Tyr Thr 1 5 90 8 PRT Artificial random library peptide 90Gln Leu Val Arg Val Ile Ser Arg 1 5 91 5 PRT Artificial random librarypeptide 91 Gln Ser Ala Asn Ile 1 5 92 4 PRT Artificial random librarypeptide 92 Gln Val Thr Lys 1 93 5 PRT Artificial random library peptide93 Arg Lys Pro Gln Lys 1 5 94 5 PRT Artificial random library peptide 94Arg Lys Ser Leu Arg 1 5 95 7 PRT Artificial random library peptide 95Arg Arg Arg Asn Phe Glu Arg 1 5 96 4 PRT Artificial random librarypeptide 96 Arg Ser Tyr Arg 1 97 7 PRT Artificial random library peptide97 Arg Thr Asn Pro Lys Val Lys 1 5 98 10 PRT Artificial random librarypeptide 98 Ser Arg Ala Ser Val Lys Gly Ser Thr Lys 1 5 10 99 6 PRTArtificial random library peptide 99 Thr Thr Arg Thr Pro Lys 1 5 100 6PRT Artificial random library peptide 100 Val Thr Pro Gln Val Lys 1 5101 8 PRT Artificial random library peptide 101 Val Val Val Glu Ser ValPro Lys 1 5 102 100 DNA Artificial synthetic sequence for peptidelibrary generation 102 nnngaattcg gacggtgccn nknnknnknn knnknnknnknnknnknnkn nknnknnknn 60 knnknnknnk nnknnknnkg gggctggaaa gcttnnnnnn 100103 21 DNA Artificial primer 103 nnnnnnaagc tttccagccc c 21 104 5 PRTArtificial random library peptide 104 Tyr Gly Pro Gln Gln 1 5 105 8 PRTArtificial random library peptide 105 Tyr Asn Ala Lys Thr Asp Arg Gly 15

1. (canceled)
 2. A process for selecting phage that are resistant to blood inactivation, comprising: a) mixing a blood component with a phage display library; and, b) selecting for phage which are resistant to inactivation by the blood component.
 3. A process for determining epitopes associated with specific parenchymal cells, comprising: a) preparing epitope display particles that are sized to exit a blood vessel and contact parenchymal cells; b) inserting the epitope display particles into a blood vessel; and, c) exposing the epitope display particles to the parenchymal cells where the epitope display particles associate with peptides specific to a cell; d) identifying cell specific epitopes.
 4. The process of claim 1 wherein selection comprises multiple rounds of selection.
 5. The process of claim 1 wherein the phage display library comprises T7 phage.
 6. The process of claim 1 wherein the blood component is mixed with the phage display library in vitro.
 7. The process of claim 1 wherein the blood component is mixed with the phage display library in vivo.
 8. The process of claim 1 wherein a variable part of the phage DNA sequence is identified.
 9. A phage that inhibits inactivation by blood components, comprising a phage having a coat peptide that protects the phage from antibody attack and inactivation.
 10. The phage of claim 8 wherein the coat peptide carboxy terminus comprises a lysine or an arginine.
 11. The phage of claim 8 wherein the peptide comprises a clone 20-6 peptide.
 12. The process of claim 1 further comprising determining phage coat peptide interactions with antibodies using the selected phage.
 13. The process of claim 11 wherein the selected phage is affinity purified.
 14. The process of claim 11 wherein a phage coat protein's sequence is determined
 15. A peptide for complexing with a drug to protect the drug from antibody inactivation during delivery, comprising determining a phage coat peptide sequence from the phage selected in claim 1 and associating the peptide with the drug to be delivered.
 16. The peptide of claim 14 wherein the peptide contains a carboxy terminal amino acid selected from the group consisting of arginine and lysine.
 17. The peptide of claim 14 wherein the peptide contains a tyrosine. 